Enhanced glucose-induced intracellular signaling promotes insulin hypersecretion: Pancreatic beta-cell functional adaptations in a model of genetic obesity and prediabetes

Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca2+ mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca2+ signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca2+ signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (BFU2013-42789-P; BFU2011-28358)This work was supported by grants from the Generalitat Valenciana (PROMETEO/2011/080)This work was supported by grants from the European Foundation for the Study Diabetes (EFSD/BI Basic Programme

Molecular mechanisms involved in the non-monotonic effect of bisphenol-a on ca2+ entry in mouse pancreatic β-cells

In regulatory toxicology, the dose-response relationship is a key element towards fulfilling safety assessments and satisfying regulatory authorities. Conventionally, the larger the dose, the greater the response, following the dogma “the dose makes the poison”. Many endocrine disrupting chemicals, including bisphenol-A (BPA), induce non-monotonic dose response (NMDR) relationships, which are unconventional and have tremendous implications in risk assessment. Although several molecular mechanisms have been proposed to explain NMDR relationships, they are largely undemonstrated. Using mouse pancreatic β-cells from wild-type and oestrogen receptor ERβ−/− mice, we found that exposure to increasing doses of BPA affected Ca2+ entry in an NMDR manner. Low doses decreased plasma membrane Ca2+ currents after downregulation of Cav2.3 ion channel expression, in a process involving ERβ. High doses decreased Ca2+ currents through an ERβ-mediated mechanism and simultaneously increased Ca2+ currents via oestrogen receptor ERα. The outcome of both molecular mechanisms explains the NMDR relationship between BPA and Ca2+ entry in β-cells.The author laboratories are funded by the Ministerio de Economía, Industria y Competitividad, Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), SAF2014-58335-P (AN) and BFU2013-42789-P (IQ) and Generalitat Valenciana, PROMETEOII/2015/016 (AN). CIBERDEM is an initiative of the Instituto de Salud Carlos III. J-A G was supported by the Robert A. Welch Foundation (E-0004)

CDK11 Promotes Cytokine-Induced Apoptosis in Pancreatic Beta Cells Independently of Glucose Concentration and Is Regulated by Inflammation in the NOD Mouse Model

Background: Pancreatic islets are exposed to strong pro-apoptotic stimuli: inflammation and hyperglycemia, during the progression of the autoimmune diabetes (T1D). We found that the Cdk11(Cyclin Dependent Kinase 11) is downregulated by inflammation in the T1D prone NOD (non-obese diabetic) mouse model. The aim of this study is to determine the role of CDK11 in the pathogenesis of T1D and to assess the hierarchical relationship between CDK11 and Cyclin D3 in beta cell viability, since Cyclin D3, a natural ligand for CDK11, promotes beta cell viability and fitness in front of glucose. Methods: We studied T1D pathogenesis in NOD mice hemideficient for CDK11 (N-HTZ), and, in N-HTZ deficient for Cyclin D3 (K11HTZ-D3KO), in comparison to their respective controls (N-WT and K11WT-D3KO). Moreover, we exposed pancreatic islets to either pro-inflammatory cytokines in the presence of increasing glucose concentrations, or Thapsigargin, an Endoplasmic Reticulum (ER)-stress inducing agent, and assessed apoptotic events. The expression of key ER-stress markers (Chop, Atf4 and Bip) was also determined. Results: N-HTZ mice were significantly protected against T1D, and NS-HTZ pancreatic islets exhibited an impaired sensitivity to cytokine-induced apoptosis, regardless of glucose concentration. However, thapsigargin-induced apoptosis was not altered. Furthermore, CDK11 hemideficiency did not attenuate the exacerbation of T1D caused by Cyclin D3 deficiency. Conclusions: This study is the first to report that CDK11 is repressed in T1D as a protection mechanism against inflammation-induced apoptosis and suggests that CDK11 lies upstream Cyclin D3 signaling. We unveil the CDK11/Cyclin D3 tandem as a new potential intervention target in T1D

Alteraciones funcionales en la célula β pancreática debidas a la exposición persistente a dosis medioambientalmente relevantes de bisfenosl-A

El bisfenol-A (BPA) es el componente principal de los plásticos de policarbonato, se usa también como aditivo en la fabricación de muchos otros tipos de plásticos y para la producción de resinas epoxi. La presencia de BPA es generalizada en multitud de productos de consumo, en contacto directo con alimentos y bebidas. El BPA ha sido detectado en la orina del 93% de los ciudadanos estadounidenses. El BPA se ha clasificado como un disruptor endocrino, que actúa como un estrógeno exógeno mediante su unión con los receptores de estrógenos ERα y ERβ. Diversos estudios epidemiológicos han relacionado la exposición a BPA con el desarrollo de ciertas patologías, incluyendo desórdenes metabólicos como la diabetes mellitus y la obesidad. Trabajos realizados durante la última década sugieren que el BPA podría contribuir en la etiología de la diabetes tipo-2, ya que el BPA causa resistencia a la insulina y altera la función de la célula β pancreática en ratones expuestos.Trabajos previos de nuestro grupo han demostrado que el BPA incrementa la expresión del gen de la insulina y que tiene un efecto insulinotrópico rápido al promover la inhibición de la actividad del canal KATP. Ambas respuestas implican la transducción de señales a través de ERα o ERβ, respectivamente, localizados fuera del núcleo. En el presente estudio, hemos estudiado cómo la exposición persistente a BPA puede modular directamente y a largo plazo la función de la célula β pancreática y el islote de Langerhans. Para ello hemos establecido un modelo de exposición in vitro utilizando islotes de Langerhans aislados o células β disgregadas que se han mantenido en presencia de BPA durante 48 horas. La caracterización de las posibles alteraciones debidas al BPA se ha realizado mediante un abordaje multidisciplinar, combinando técnicas electrofisiológicas (actividad eléctrica, corrientes macroscópicas y medidas de la capacidad de membrana), microscopía de fluorescencia con sondas sensibles a calcio, medida de la secreción de insulina y estudio de los cambios en la expresión de ARNm mediante PCR a tiempo real. Los resultados que hemos obtenido muestran que la exposición a BPA provoca cambios muy significativos en el funcionamiento de la célula β pancreática. A una dosis tan baja como 1 nM, el BPA altera la forma del potencial de acción de las células β en respuesta a glucosa, disminuyendo su amplitud y haciéndolo más ancho. A esta dosis de exposición, hallamos que la entrada de calcio, en respuesta a glucosa o estímulos despolarizantes no-metabólicos, estaba disminuida en células β expuestas. Sin embargo, al incrementar la dosis de exposición a 100 nM BPA, los cambios en la entrada de calcio no se detectaron, por lo que la relación entre la dosis de BPA y el efecto que describimos se produce de un modo no monotónico. El fenómeno de la nomonotonicidad aparece frecuentemente en la literatura en relación a los efectos provocados por la exposición a EDCs. Así mismo, la exposición a 1 nM BPA altera muy significativamente la capacidad exocitótica de las células β y su secreción deinsulina. La disección farmacológica de la corriente macroscópica de calcio revela que la reducción particular de la corriente tipo-R era la responsable de este cambio. Empleando PCR a tiempo real, hallamos que la transcripción del canal Cv2.3, que conduce la corriente R, estaba reducida en las células cultivadas en presencia de BPA. Este efecto transcripcional se produjo por igual con independencia de la dosis de BPA empleada, lo que indica una relación monotónica entre la dosis de BPA y su efecto sobre la expresión del canal, a pesar de no observarse un efecto de BPA sobre la entrada de calcio a las dosis más altas que utilizamos. El efecto de una dosis baja de BPA sobre la entrada de calcio en la célula β se reprodujo al emplear un agonista específico de ERβ (DPN), mientras que fueron inexistentes en células β provenientes de ratones ERβ-knockout. Estos resultados parecen indicar que ERβ está implicado en la mediación de las respuestas a dosis bajas de BPA (1 nM). Sin embargo, la discrepancia entre la nomonotonicidad en la entrada de calcio y la monotonicidad en el efecto sobre la expresión del canal tipo-R, sugiere la existencia de otro mecanismo modulado por dosis más altas de BPA que actúa regulando la entrada de calcio de forma opuesta. Nuestros resultados sugieren que la activación de ERα a dosis altas (100 nM y 1 μM) provoca la potenciación de la corriente macroscópica de calcio, y que este mecanismo es dependiente de la activación de la vía de PI3K. El solapamiento de estos efectos opuestos a través de ERβ y ERα tendría como consecuencia la aparición de la nomonotonicidad.Bisphenol-A (BPA) is the main component of the polycarbonate plastics, and it is also used as an additive in the manufacture of many others types of plastics and epoxy resins. BPA is extensively present in daily consumer goods in close contact with food and beverages, and it is detected in the urine of 93% of USA citizens. BPA has been classified as an endocrine disruptor chemical (EDC), which acts like a xenoestrogen by binding to estrogen receptors ERα and ERβ. Epidemiological studies show that BPA exposure is linked to the development of some diseases, including metabolic disorders like diabetes mellitus and obesity. In the last decade, several works suggest that BPA could be involved in the etiology of type-2 diabetes, because it elicits insulin resistance and alters pancreatic β-cell function in BPA-treated mice. Previous works of our group have demonstrated that BPA increases the expression levels of the insulin gene, and that BPA-induced insulinotropic effects depends on rapid KATP inhibition. These mechanisms are mediated by ERα and ERβ extranuclear-initiated cascades respectively. The main goal of this project is to characterize how BPA chronic exposure could directly modulate the function of pancreatic β-cells and islets of Langerhans. To address this aim, we have established an in vitro long-lasting exposure model using dispersed β-cells and isolated islets of Langerhans, both maintained during 48 hours with BPA always present in the culture medium. We have used a multidisciplinary approach combining several experimental techniques: electrophysiological measurements (electrical activity, global currents and changes in membrane capacity), intracellular calcium recordings using fluorescent dyes, insulin secretion and mRNA expression levels using qPCR. Our results demonstrate that BPA causes significant alterations in β-cell function. BPA at a dose as low as 1 nM promotes changes in the shape of glucose-induced action potentials, decreasing its amplitude and increasing its width. Likewise, 1 nM BPA reduces calcium entry triggered by membrane depolarizations, both in response to glucose or non-metabolic stimuli. Nevertheless, a higher dose, 100 nM BPA, has no effect on our endpoint. Accordingly, we conclude that the relationship between BPA and its effects on calcium entry is non-monotonic. Nonmonotonicity is a common phenomenon in the field of EDCs. The exposure to 1 nM BPA alters in the same extent the exocytotic capacity and insulin secretion of pancreatic β-cells. Pharmacological dissection of macroscopic calcium current suggests that BPA-induced decrease on calcium influx is a consequence of R-type calcium current reduction. Using qPCR, we found that BPA reduces mRNA expression levels of R-type pore-forming subunit, and this transcriptional effect is produced by BPA at any tested doses, revealing a monotonic relationship at this regulation level. The effect of BPA on calcium entry is mimicked using a specific ERβ agonist (DPN), and abolished in β-cells from ERβ knockout mice. These findings suggest that the functional alterations promoted by low doses of BPA are mediated predominantly by ERβ. Disagreement between BPA-induced effects on calcium entry and transcriptional regulation of R-type calcium channel suggests the participation of a counteracting mechanism regulated by higher BPA doses. We found that high BPA doses (≥ 100 nM) potentiate voltage-gated calcium entry in pancreatic β-cells through extranuclear ERα-mediated PI3K-dependent mechanism. We conclude that the NMDR relationship between BPA exposure and calcium entry seems to be the outcome of the opposite effects mediated by ERβ and ERα on the processes regulating voltage-operated calcium currents in pancreatic β-cells

Extranuclear-initiated estrogenic actions of endocrine disrupting chemicals: Is there toxicology beyond paracelsus?

Endocrine Disrupting Chemicals (EDCs), including bisphenol-A (BPA) do not act as traditional toxic chemicals inducing massive cell damage or death in an unspecific manner. EDCs can work upon binding to hormone receptors, acting as agonists, antagonists or modulators. Bisphenol-A displays estrogenic activity and, for many years it has been classified as a weak estrogen, based on the classic transcriptional action of estrogen receptors serving as transcription factors. However, during the last two decades our knowledge about estrogen signaling has advanced considerably. It is now accepted that estrogen receptors ERα and ERβ activate signaling pathways outside the nucleus which may or may not involve transcription. In addition, a new membrane estrogen receptor, GPER, has been proposed. Pharmacological and molecular evidence, along with results obtained in genetically modified mice, demonstrated that BPA, and its substitute BPS, are potent estrogens acting at nanomolar concentrations via extranuclear ERα, ERβ, and GPER. The different signaling pathways activated by BPA and BPS explain the well-known estrogenic effects of low doses of EDCs as well as non-monotonic dose-response relationships. These signaling pathways may help to explain the actions of EDCs with estrogenic activity in the etiology of different pathologies, including type-2 diabetes and obesity.The author laboratories are funded by the Ministerio de Economía y Competitividad (SAF2014-58335-P and BFU2013-42789-P) and Generalitat Valenciana (PROMETEOII/2015/016). CIBERDEM is an initiative of the Instituto de Salud Carlos III

Enhanced glucose-induced intracellular signaling promotes insulin hypersecretion: pancreatic beta-cell functional adaptations in a model of genetic obesity and prediabetes

sem informaçãoObesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca(2+) mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca(2+) signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca(2+) signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported4044655sem informaçãosem informaçãosem informaçã

Oestrogen receptor β mediates the actions of bisphenol-A on ion channel expression in mouse pancreatic beta cells

Aims/hypothesis Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical that has been associated with type 2 diabetes development. Low doses of BPA modify pancreatic beta cell function and induce insulin resistance; some of these effects are mediated via activation of oestrogen receptors α (ERα) and β (ERβ). Here we investigated whether low doses of BPA regulate the expression and function of ion channel subunits involved in beta cell function. Methods Microarray gene profiling of isolated islets from vehicle- and BPA-treated (100 μg/kg per day for 4 days) mice was performed using Affymetrix GeneChip Mouse Genome 430.2 Array. Expression level analysis was performed using the normalisation method based on the processing algorithm ‘robust multi-array average’. Whole islets or dispersed islets from C57BL/6J or oestrogen receptor β (ERβ) knockout (Erβ−/−) mice were treated with vehicle or BPA (1 nmol/l) for 48 h. Whole-cell patch-clamp recordings were used to measure Na+ and K+ currents. mRNA expression was evaluated by quantitative real-time PCR. Results Microarray analysis showed that BPA modulated the expression of 1440 probe sets (1192 upregulated and 248 downregulated genes). Of these, more than 50 genes, including Scn9a, Kcnb2, Kcnma1 and Kcnip1, encoded important Na+ and K+ channel subunits. These findings were confirmed by quantitative RT-PCR in islets from C57BL/6J BPA-treated mice or whole islets treated ex vivo. Electrophysiological measurements showed a decrease in both Na+ and K+ currents in BPA-treated islets. The pharmacological profile indicated that BPA reduced currents mediated by voltage-activated K+ channels (Kv2.1/2.2 channels) and large-conductance Ca2+-activated K+ channels (KCa1.1 channels), which agrees with BPA’s effects on gene expression. Beta cells from ERβ−/− mice did not present BPA-induced changes, suggesting that ERβ mediates BPA’s effects in pancreatic islets. Finally, BPA increased burst duration, reduced the amplitude of the action potential and enlarged the action potential half-width, leading to alteration in beta cell electrical activity. Conclusions/interpretation Our data suggest that BPA modulates the expression and function of Na+ and K+ channels via ERβ in mouse pancreatic islets. Furthermore, BPA alters beta cell electrical activity. Altogether, these BPA-induced changes in beta cells might play a role in the diabetogenic action of BPA described in animal models.This work was supported by Ministerio de Economía y Competitividad, Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) grants BPU2017-86579-R and SAF2014-58335-P (AN) and BFU2016-77125-R (IQ); Generalitat Valenciana PROMETEO II/2015/016 (AN); Grant for Networks of Excellence from MICINN ‘Nuclear Receptors in Cancer, Metabolism and Inflammation’ (NuRCaMeIn) (SAF2017-90604-REDT); Spanish Institute of Health Carlos III grants PI16/00259 (AH), PI17/02104 and RD16/0011/0034 (BS) co-financed by FEDER Funds. LM holds a Juan de la Cierva fellowship from the Ministry of Economy, Industry and Competitiveness (IJCI-2015-24482). CIBERDEM is an initiative of the Instituto de Salud Carlos III. J-AG was supported by the Robert A.Welch Foundation (E-0004)

Cortistatin regulates glucose-induced electrical activity and insulin secretion in mouse pancreatic beta-cells

Although there is growing evidence that cortistatin regulates several functions in different tissues, its role in the endocrine pancreas is not totally known. Here, we aim to study the effect of cortistatin on pancreatic beta-cells and glucose-stimulated insulin secretion (GSIS). Exposure of isolated mouse islets to cortistatin inhibited GSIS. This effect was prevented using a somatostatin receptor antagonist. Additionally, cortistatin hyperpolarized the membrane potential and reduced glucose-induced action potentials in isolated pancreatic beta-cells. Cortistatin did not modify ATP-dependent K+ (KATP) channel activity. In contrast, cortistatin increased the activity of a small conductance channel with characteristics of G protein-coupled inwardly rectifying K+ (GIRK) channels. The cortistatin effects on membrane potential and GSIS were largely reduced in the presence of a GIRK channel antagonist and by down-regulation of GIRK2 with small interfering RNA. Thus, cortistatin acts as an inhibitory signal for glucose-induced electrical activity and insulin secretion in the mouse pancreatic beta-cell.This work was supported by grants from the Ministerio de Economía, Industria y Competitividad (Spain), Agencia Estatal de Investigación (Spain) and Fondo Europeo de Desarrollo Regional (BFU2013-42789; BFU2017-86579-R; BFU2016-77125-R), Generalitat Valenciana (Spain, PROMETEOII/2015/016), CNPq (Brazil; 302261/2014-1; 306359/2017-0) and Junta de Andalucía (Spain; CTS-1406, BIO-0139). L.M. holds a Juan de la Cierva fellowship from the Ministry of Economy, Industry and Competitiveness (Spain; IJCI-2015-24482). CIBERDEM and CIBERobn are an initiative of the Instituto de Salud Carlos III (Spain)

Cortistatin regulates glucose-induced electrical activity and insulin secretion in mouse pancreatic beta-cells

Although there is growing evidence that cortistatin regulates several functions in different tissues, its role in the endocrine pancreas is not totally known. Here, we aim to study the effect of cortistatin on pancreatic beta-cells and glucose-stimulated insulin secretion (GSIS). Exposure of isolated mouse islets to cortistatin inhibited GSIS. This effect was prevented using a somatostatin receptor antagonist Additionally, cortistatin hyperpolarized the membrane potential and reduced glucose-induced action potentials in isolated pancreatic beta-cells. Cortistatin did not modify ATP-dependent K+ (K-ATP) channel activity. In contrast, cortistatin increased the activity of a small conductance channel with characteristics of G protein-coupled inwardly rectifying K+ (GIRK) channels. The cortistatin effects on membrane potential and GSIS were largely reduced in the presence of a GIRK channel antagonist and by down-regulation of GIRK2 with small interfering RNA. Thus, cortistatin acts as an inhibitory signal for glucose-induced electrical activity and insulin secretion in the mouse pancreatic beta-cell479123132CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ302261/2014-1; 306359/2017-0We thank M.S. Ramon and M.L. Navarro for their expert technical assistance, and R. Pérez-Rodríguez for her help with English. This work was supported by grants from the Ministerio de Economía, Industria y Competitividad (Spain), Agencia Estatal de Investigación (Spain) and Fondo Europeo de Desarrollo Regional (BFU2013-42789; BFU2017-86579-R; BFU2016-77125-R), Generalitat Valenciana (Spain, PROMETEOII/2015/016), CNPq (Brazil; 302261/2014-1; 306359/2017-0) and Junta de Andalucía (Spain; CTS-1406, BIO-0139). L.M. holds a Juan de la Cierva fellowship from the Ministry of Economy, Industry and Competitiveness (Spain; IJCI-2015-24482). CIBERDEM and CIBERobn are an initiative of the Instituto de Salud Carlos III (Spain