Exposure to glyphosate-based herbicides (GBH) may affect metabolism and induce endocrine alterations. This study evaluated whether environmentally relevant low doses of GBH affect glucose homeostasis during pregnancy and the metabolism of offspring later in life. Pregnant Wistar rats received daily oral doses of either 0.5 or 50 mg/kg of GBH or water (control) throughout the gestation period. No significant changes were observed in biometric and metabolic parameters during pregnancy. However, hepatic alterations were detected in GBH-treated rats, including reduced glycogen content and decreased global genome methylation. Offspring exhibited a transient delay in neurodevelopmental reflexes. Body mass and food intake remained unchanged up to postnatal day 70. After this period, animals were subjected to a metabolic challenge with dexamethasone. While dexamethasone-treated animals showed metabolic alterations typical of glucocorticoid exposure (glucose intolerance, insulin insensitivity, dyslipidemia), maternal GBH exposure during pregnancy did not exacerbate these effects in the offspring. Additionally, changes in DNA methylation-related gene expression were observed in female but not male offspring. In vitro, GBH exhibited greater cytotoxicity than glyphosate alone, significantly reducing the viability of 3T3-L1 adipocytes and HepG2 hepatocytes, whereas glyphosate alone had no significant effects, even at higher concentrations. Overall, gestational exposure to GBH under regulatory safe doses does not cause substantial metabolic disturbances in dams or their offspring. However, the existence of alterations in hepatic glucose metabolism and effects on hepatic epigenetic regulation at these low doses, along with the cytotoxic in vitro effects of GBH at high concentrations, warrants attention regarding human exposure and health impacts.

Background: Gestational diabetes mellitus (GDM) is the most common metabolic disease during pregnancy and increases the prevalence of type 2 diabetes in both mothers and children. GDM management provides an opportunity to prevent and lower the global burden of diabetes across life. Molecular mechanisms underlying GDM are not completely understood. In this study, we explore the role of transforming growth factor beta (TGF-(3) signaling in GDM, as this pathway reportedly affects pancreatic (3-cell development, function, and proliferation. Methods: We developed a GDM animal model. Serum circulating levels of TGF-(3 family ligands were measured in mice and human GDM. Pancreatic TGF-(3 signaling was investigated via gene and protein expression. Results: Our GDM animal model recapitulates the main pathophysiological features of human GDM, including glucose intolerance, decreased insulin sensitivity and pancreatic (3-cell malfunction. Islets from GDM mice showed impaired insulin secretion and content, altered ion channel activity, and decreased (3-cell replication rate. This was accompanied by increased Smad2 signaling activation. Elevated serum activin-A and inhibin levels were found in mice and human GDM, suggesting their role as upstream signaling transducers of pancreatic Smad2 activation. Pharmacological inhibition of TGF-(3/Activin-Smad2 signaling in mouse pancreatic islets resulted in improved pancreatic (3-cell function and regeneration capacity. Conclusions: Our data suggest that disruption of the pancreatic Smad2 pathway plays a critical role in the pathogenesis of GDM, contributing to abnormal glucose homeostasis and inadequate insulin secretion. Attenuation of this signaling pathway may represent a putative therapeutic target for GDM.

Diabetes mellitus has become one of the most devastating metabolic disorders of modern times. The causes behind the alarming increase in diabetes incidence are known to be multifactorial with exposure to some environmental chemicals as one important contributing factor. In the present article we will describe the most relevant effects of some widespread environmental chemicals on pancreatic β-cell function and thus on glucose homeostasis. Knowledge of environmental disruptors of β-cell physiology may contribute to a better understanding of the role of these compounds in the onset of diabetes.

The global prevalence of type 2 diabetes (T2D) continues to rise, and predictions indicate alarming records in coming decades. Although pancreatic β-cell dysfunction and insulin resistance are key factors in the etiology of T2D, the impairment of α-cells has been also implicated. Hyperglucagonemia and altered suppression of glucagon release can be frequently found in individuals with T2D, contributing to hyperglycemia. Bile acids have emerged as novel signaling molecules that regulate metabolism. A wealth of evidence shows that oral treatment with the bile acid TUDCA has therapeutic benefits in T2D, primarily by improving both insulin release from β-cells and insulin sensitivity in peripheral tissues. However, it is unknown whether TUDCA could affect other processes involved in the control of glucose metabolism. Here, we show that acute administration of TUDCA exerts a glucagonstatic action on mouse pancreatic islets and glucagon-releasing αTC1-9 cells. Pharmacological and/or molecular inhibition of the sphingosine-1-phosphate receptor 2 (S1PR2) and the PI3K pathway blunted the TUDCA effect on glucagon release. Additionally, TUDCA increased the activity of ATP-sensitive K+ (KATP) channels, decreased action currents and inhibited Ca2+ signaling in α-cells without directly affecting the exocytotic process. Glucose-induced suppression of glucagon secretion was found to be compromised under hyperglycemic conditions, yet TUDCA was able to inhibit α-cell function, highlighting its glucagonstatic effect in a pathological context. Collectively, these findings suggest that TUDCA-induced inhibition of glucagon secretion involves the opening of α-cell KATP channels and activation of the S1PR2/PI3K pathway, expanding the repertoire of potential therapeutic benefits of TUDCA in diabetes treatment.

BACKGROUND:Gestational diabetes mellitus (GDM) is the most common metabolic disease during pregnancy and increases the prevalence of type 2 diabetes in both mothers and children. GDM management provides an opportunity to prevent and lower the global burden of diabetes across life. Molecular mechanisms underlying GDM are not completely understood. In this study, we explore the role of transforming growth factor beta (TGF-β) signaling in GDM, as this pathway reportedly affects pancreatic β-cell development, function, and proliferation. METHODS:We developed a GDM animal model. Serum circulating levels of TGF-β family ligands were measured in mice and human GDM. Pancreatic TGF-β signaling was investigated via gene and protein expression. RESULTS:Our GDM animal model recapitulates the main pathophysiological features of human GDM, including glucose intolerance, decreased insulin sensitivity and pancreatic β-cell malfunction. Islets from GDM mice showed impaired insulin secretion and content, altered ion channel activity, and decreased β-cell replication rate. This was accompanied by increased Smad2 signaling activation. Elevated serum activin-A and inhibin levels were found in mice and human GDM, suggesting their role as upstream signaling transducers of pancreatic Smad2 activation. Pharmacological inhibition of TGF-β/Activin-Smad2 signaling in mouse pancreatic islets resulted in improved pancreatic β-cell function and regeneration capacity. CONCLUSIONS:Our data suggest that disruption of the pancreatic Smad2 pathway plays a critical role in the pathogenesis of GDM, contributing to abnormal glucose homeostasis and inadequate insulin secretion. Attenuation of this signaling pathway may represent a putative therapeutic target for GDM.

Hyperglucagonemia has been implicated in the pathogenesis of type 2 diabetes (T2D). In contrast to β-cells, studies on the function of the pancreatic α-cell in T2D are scarce. Consequently, the processes underlying hyperglucagonemia and α-cell dysfunction are largely unknown, limiting the appropriate design of specific pharmacological and therapeutic strategies. In the current study, we aimed to analyze the alterations of the pancreatic α-cell and its glucagon responses in diabetic db/db mice at early stages of the disease. In this context of glucose intolerance, hyperinsulinemia, and β-cell dysfunction, hyperglucagonemia was only present at fed conditions and was associated with insulin resistance. Yet, we found that the glucagon-to-insulin ratio in db/db mice did not change with fed or fasted states, further supporting that the metabolic regulation of glucagon release was impaired. Pancreatic β-cell dysfunction in db/db mice was manifested by increased basal secretion from isolated islets along with reduced insulin content. In contrast, α-cells from diabetic animals presented upregulated secretion and islet content of glucagon compared with controls. Electrophysiological analysis of dispersed α-cells revealed that altered secretion was not the result of impaired exocytosis. Instead, we found defective regulation of Ca2+ signaling by glucose. Besides these functional alterations, we also observed augmented α-cell mass in diabetic mice, which was accompanied by disrupted islet cytoarchitecture as well as increased α-cell size and number, without pieces of evidence of upregulated proliferation. Overall, these findings indicate that hyperglucagonemia in early T2D results from multifaceted α-cell deregulation in mice.

Pancreatic β cell dysfunction is a key feature of type 2 diabetes, and novel regulators of insulin secretion are desirable. Here, we report that succinate receptor 1 (SUCNR1) is expressed in β cells and is upregulated in hyperglycemic states in mice and humans. We found that succinate acted as a hormone-like metabolite and stimulated insulin secretion via a SUCNR1-Gq-PKC-dependent mechanism in human β cells. Mice with β cell-specific Sucnr1 deficiency exhibited impaired glucose tolerance and insulin secretion on a high-fat diet, indicating that SUCNR1 is essential for preserving insulin secretion in diet-induced insulin resistance. Patients with impaired glucose tolerance showed an enhanced nutrition-related succinate response, which correlates with the potentiation of insulin secretion during intravenous glucose administration. These data demonstrate that the succinate/SUCNR1 axis is activated by high glucose and identify a GPCR-mediated amplifying pathway for insulin secretion relevant to the hyperinsulinemia of prediabetic states.

Humans are constantly exposed to many environmental pollutants, some of which have been largely acknowledged as key factors in the development of metabolic disorders such as diabetes and obesity. These chemicals have been classified as endocrine-disrupting chemicals (EDCs) and, more recently, since they can interfere with metabolic functions, they have been renamed as metabolism-disrupting chemicals (MDCs). MDCs are present in many consumer products, including food packaging, personal care products, plastic bottles and containers, and detergents. The scientific literature has ever-increasingly focused on insulin-releasing pancreatic β-cells as one of the main targets for MDCs. Evidence highlights that these substances may disrupt glucose homeostasis by altering pancreatic β-cell physiology. However, their potential impact on glucagon-secreting pancreatic α-cells remains poorly known despite the essential role that this cellular type plays in controlling glucose metabolism. In the present study, we have selected seven paradigmatic MDCs representing major toxic classes, including bisphenols, phthalates, perfluorinated compounds, metals, and pesticides. By using an in vitro cell-based model, the pancreatic α-cell line αTC1-9, we have explored the effects of these compounds on pancreatic α-cell viability, gene expression, and secretion. We found that cell viability was moderately affected after bisphenol-A (BPA), bisphenol-F (BPF), and perfluorooctanesulfonic acid (PFOS) exposure, although cytotoxicity was relatively low. In addition, all bisphenols, as well as di(2-ethylhexyl) phthalate (DEHP) and cadmium chloride (CdCl2), promoted a marked decreased on glucagon secretion, together with changes in the expression of glucagon and/or transcription factors involved in cell function and identity, such as Foxo1 and Arx. Overall, our results indicated that most of the selected chemicals studied caused functional alterations in pancreatic α-cells. Moreover, we revealed, for the first time, their direct effects on key molecular aspects of pancreatic α-cell biology.

Plastic pollution breaks a planetary boundary threatening wildlife and humans through its physical and chemical effects. Of the latter, the release of endocrine disrupting chemicals (EDCs) has consequences on the prevalence of human diseases related to the endocrine system. Bisphenols (BPs) and phthalates are two groups of EDCs commonly found in plastics that migrate into the environment and make low-dose human exposure ubiquitous. Here we review epidemiological, animal, and cellular studies linking exposure to BPs and phthalates to altered glucose regulation, with emphasis on the role of pancreatic β-cells. Epidemiological studies indicate that exposure to BPs and phthalates is associated with diabetes mellitus. Studies in animal models indicate that treatment with doses within the range of human exposure decreases insulin sensitivity and glucose tolerance, induces dyslipidemia, and modifies functional β-cell mass and serum levels of insulin, leptin, and adiponectin. These studies reveal that disruption of β-cell physiology by EDCs plays a key role in impairing glucose homeostasis by altering the mechanisms used by β-cells to adapt to metabolic stress such as chronic nutrient excess. Studies at the cellular level demonstrate that BPs and phthalates modify the same biochemical pathways involved in adaptation to chronic excess fuel. These include changes in insulin biosynthesis and secretion, electrical activity, expression of key genes, and mitochondrial function. The data summarized here indicate that BPs and phthalates are important risk factors for diabetes mellitus and support a global effort to decrease plastic pollution and human exposure to EDCs.

Type 2 diabetes is characterised by hyperglucagonaemia and perturbed function of pancreatic glucagon-secreting alpha cells but the molecular mechanisms contributing to these phenotypes are poorly understood. Insulin-degrading enzyme (IDE) is present within all islet cells, mostly in alpha cells, in both mice and humans. Furthermore, IDE can degrade glucagon as well as insulin, suggesting that IDE may play an important role in alpha cell function in vivo. We have generated and characterised a novel mouse model with alpha cell-specific deletion of Ide, the A-IDE-KO mouse line. Glucose metabolism and glucagon secretion in vivo was characterised; isolated islets were tested for glucagon and insulin secretion; alpha cell mass, alpha cell proliferation and α-synuclein levels were determined in pancreas sections by immunostaining. Targeted deletion of Ide exclusively in alpha cells triggers hyperglucagonaemia and alpha cell hyperplasia, resulting in elevated constitutive glucagon secretion. The hyperglucagonaemia is attributable in part to dysregulation of glucagon secretion, specifically an impaired ability of IDE-deficient alpha cells to suppress glucagon release in the presence of high glucose or insulin. IDE deficiency also leads to α-synuclein aggregation in alpha cells, which may contribute to impaired glucagon secretion via cytoskeletal dysfunction. We showed further that IDE deficiency triggers impairments in cilia formation, inducing alpha cell hyperplasia and possibly also contributing to dysregulated glucagon secretion and hyperglucagonaemia. We propose that loss of IDE function in alpha cells contributes to hyperglucagonaemia in type 2 diabetes.

Aging is associated with a decline in peripheral insulin sensitivity and an increased risk of impaired glucose tolerance and type 2 diabetes. During conditions of reduced insulin sensitivity, pancreatic β cells undergo adaptive responses to increase insulin secretion and maintain euglycemia. However, the existence and nature of β-cell adaptations and/or alterations during aging are still a matter of debate. In this study, we investigated the effects of aging on β-cell function from control (3-month-old) and aged (20-month-old) mice. Aged animals were further categorized into 2 groups: high insulin sensitive (aged-HIS) and low insulin sensitive (aged-LIS). Aged-LIS mice were hyperinsulinemic, glucose intolerant, and displayed impaired glucose-stimulated insulin and C-peptide secretion, whereas aged-HIS animals showed characteristics in glucose homeostasis similar to controls. In isolated β cells, we observed that glucose-induced inhibition of KATP channel activity was reduced with aging, particularly in the aged-LIS group. Glucose-induced islet NAD(P)H production was decreased in aged mice, suggesting impaired mitochondrial function. In contrast, voltage-gated Ca2+ currents were higher in aged-LIS β cells, and pancreatic islets of both aged groups displayed increased glucose-induced Ca2+ signaling and augmented insulin secretion compared with controls. Morphological analysis of pancreas sections also revealed augmented β-cell mass with aging, especially in the aged-LIS group, as well as ultrastructural β-cell changes. Altogether, these findings indicate that aged mouse β cells compensate for the aging-induced alterations in the stimulus-secretion coupling, particularly by adjusting their Ca2+ influx to ensure insulin secretion. These results also suggest that decreased peripheral insulin sensitivity exacerbates the effects of aging on β cells.

The prevalence of type 2 diabetes (T2D) and impaired glucose tolerance (IGT) increases with ageing. T2D generally results from progressive impairment of the pancreatic islets to adapt β-cell mass and function in the setting of insulin resistance and increased insulin demand. Several studies have shown an age-related decline in peripheral insulin sensitivity. However, a precise understanding of the pancreatic β-cell response in ageing is still lacking. In this review, we summarize the age-related alterations, adaptations and/or failures of β-cells at the molecular, morphological and functional levels in mouse and human. Age-associated alterations include processes such as β-cell proliferation, apoptosis and cell identity that can influence β-cell mass. Age-related changes also affect β-cell function at distinct steps including electrical activity, Ca2+ signaling and insulin secretion, among others. We will consider the potential impact of these alterations and those mediated by senescence pathways on β-cells and their implications in age-related T2D. Finally, given the great diversity of results in the field of β-cell ageing, we will discuss the sources of this heterogeneity. A better understanding of β-cell biology during ageing, particularly at older ages, will improve our insight into the contribution of β-cells to age-associated T2D and may boost new therapeutic strategies.

Second-generation antipsychotic (SGA) drugs have been associated with the development of type 2 diabetes and the metabolic syndrome in patients with schizophrenia. In this study, we aimed to investigate the effects of two different SGA drugs, olanzapine and aripiprazole, on metabolic state and islet function and plasticity. We analysed the functional adaptation of beta cells in 12-week-old B6;129 female mice fed an olanzapine- or aripiprazole-supplemented diet (5.5–6.0 mg kg−1 day−1) for 6 months. Glucose and insulin tolerance tests, in vivo glucose-stimulated insulin secretion and indirect calorimetry were performed at the end of the study. The effects of SGAs on beta cell plasticity and islet serotonin levels were assessed by transcriptomic analysis and immunofluorescence. Insulin secretion was assessed by static incubations and Ca2+ fluxes by imaging techniques. Treatment of female mice with olanzapine or aripiprazole for 6 months induced weight gain (p<0.01 and p<0.05, respectively), glucose intolerance (p<0.01) and impaired insulin secretion (p<0.05) vs mice fed a control chow diet. Aripiprazole, but not olanzapine, induced serotonin production in beta cells vs controls, likely by increasing tryptophan hydroxylase 1 (TPH1) expression, and inhibited Ca2+ flux. Of note, aripiprazole increased beta cell size (p<0.05) and mass (p<0.01) vs mice fed a control chow diet, along with activation of mechanistic target of rapamycin complex 1 (mTORC1)/S6 signalling, without preventing beta cell dysfunction. Both SGAs induced weight gain and beta cell dysfunction, leading to glucose intolerance; however, aripiprazole had a more potent effect in terms of metabolic alterations, which was likely a result of its ability to modulate the serotonergic system. The deleterious metabolic effects of SGAs on islet function should be considered while treating patients as these drugs may increase the risk for development of the metabolic syndrome and diabetes.

Bisphenol-S (BPS) and Bisphenol-F (BPF) are current Bisphenol-A (BPA) substitutes. Here we used pancreatic β-cells from wild type (WT) and estrogen receptor β (ERβ) knockout (BERKO) mice to investigate the effects of BPS and BPF on insulin secretion, and the expression and activity of ion channels involved in β-cell function. BPS or BPF rapidly increased insulin release and diminished ATP-sensitive K+ (KATP) channel activity. Similarly, 48 h treatment with BPS or BPF enhanced insulin release and decreased the expression of several ion channel subunits in β-cells from WT mice, yet no effects were observed in cells from BERKO mice. PaPE-1, a ligand designed to preferentially trigger extranuclear-initiated ER pathways, mimicked the effects of bisphenols, suggesting the involvement of extranuclear-initiated ERβ pathways. Molecular dynamics simulations indicated differences in ERβ ligand-binding domain dimer stabilization and solvation free energy among different bisphenols and PaPE-1. Our data suggest a mode of action involving ERβ whose activation alters three key cellular events in β-cell, namely ion channel expression and activity, and insulin release. These results may help to improve the hazard identification of bisphenols.

Abstract Bisphenol-A (BPA) is one of the highest volume chemicals produced worldwide. It is used as the base compound in the manufacture of polycarbonate plastics, epoxies and resins. Humans are consistently exposed to BPA and consistently it has been detected in the majority of individuals examined. Experimental research in animals, as well as human epidemiological studies, converge to conclude that BPA is a risk factor for the development of type 2 diabetes. In previous studies we have demonstrated that the exposure to BPA during embryonic development promote an increment of pancreatic β-cell mass. This was correlated with increased β-cell division and altered global gene expression in pancreatic β-cells. The aim of this work was to determinate whether ERβ was involved in the in the β-cell mass and proliferation increment observed in male mice offspring. ERβ+/- pregnant mice were treated with vehicle or BPA (10 μg/kg/day) from day 9 to 16 of gestation. Offspring pancreatic β-cell mass was measured at postnatal day 0 (P0) and 30 (P30). For ex vivo experiments Wild-type (WT) and ERβ-/- neonates as well as adult male and female mice were used. For in vitro, single islets cells were cultured for 48 h in the presence of 10 μmol/L BrdU, and vehicle, BPA (1, 10, 100 nM) or the specific ERβ agonist WAY200070 (1, 10, 100 nM). β-cell proliferation rate was quantified as the percentage of BrdU-positive pancreatic β-cells. In vivo exposure to BPA during pregnancy promoted an increment of pancreatic β-cell mass and proliferation in WT mice at P30 which was absent in ERβ -/- mice. In order to explore if these changes were related to a direct action of BPA on pancreatic β-cell division we performed a series of ex vivo experiments. Augmented β-cell proliferation rate was observed in BPA-exposed β-cells isolated from both adult male and female WT animals in comparison to controls. The increment was significant at all BPA doses tested. The effect was imitated by the selective ERβ agonist, WAY200070, and was abolished in cells from ERβ-/- mice. We also explored the effects of BPA in pancreatic β-cells from neonates and found an increment in BPA-exposed cells compared to controls, although the difference was only significant at the dose of 1 nM. A similar effect was observed in neonate cells treated with WAY200070 (10 nM). The effects on β-cell replication were abolished in cells from ERβ-/- neonate mice treated either with BPA or WAY200070. Our findings suggest that BPA modulate pancreatic β-cell growth and mass in an ERβ-dependent manner. This could have important implications for metabolic programming of T2DM. Ministerio de Economía y Competitividad, Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) grants BPU2017-86579-R (AN) and BFU2016-77125-R (IQ); Generalitat Valenciana PROMETEO II/2015/016 (AN). CIBERDEM is an initiative of the Instituto de Salud Carlos III.

Diabetes (T2DM) is a major global health issue, and developing new approaches to its prevention is of paramount importance. We hypothesized that abnormalities in lipid metabolism are involved in alpha-cell deregulation. We therefore studied the metabolic factors underlying alpha-cell dysfunction in T2DM progression after a dietary intervention (Mediterranean and low-fat). Additionally, we evaluated whether postprandial glucagon levels may be considered as a predictive factor of T2DM in cardiovascular patients. Non-T2DM participants from the CORDIOPREV study were categorized by tertiles of the area under the curve (AUC) for triacylglycerols, and also by tertiles of AUC for glucagon. Our results showed that patients with higher triacylglycerols levels presented elevated postprandial glucagon (P = 0.009). Moreover, we observed higher risk of T2DM (Hazard ratio: 2.65; 95% confidence interval: 1.56-4.53) in subjects with elevated glucagon. In conclusion, high postprandial lipemia may induce alpha-cell dysfunction in cardiovascular patients. Our results also showed that postprandial glucagon levels could be used to predict T2DM development.

Bisphenol-A (BPA) is a widespread endocrine disrupting chemical that constitutes a risk factor for type 2 diabetes mellitus (T2DM). Data from animal and human studies have demonstrated that early exposure to BPA results in adverse metabolic outcomes in adult life. In the present work, we exposed pregnant heterozygous estrogen receptor β (ERβ) knock out (BERKO) mice to 10 μg/kg/day BPA, during days 9-16 of pregnancy, and measured β-cell mass and proliferation in wildtype (WT) and BERKO male offspring at postnatal day 30. We observed increased pancreatic β-cell proliferation and mass in WT, yet no effect was produced in BERKO mice. Dispersed islet cells in primary culture treated with 1 nM BPA showed an enhanced pancreatic β-cell replication rate, which was blunted in pancreatic β-cells from BERKO mice and mimicked by the selective ERβ agonist WAY200070. This increased β-cell proliferation was found in male adult as well as in neonate pancreatic β-cells, suggesting that BPA directly impacts β-cell division at earliest stages of life. These findings strongly indicate that BPA during pregnancy upregulates pancreatic β-cell division and mass in an ERβ-dependent manner. Thus, other natural or artificial chemicals may use this ERβ-mediated pathway to promote similar effects.

BACKGROUND:Pregnancy represents a major metabolic challenge for the mother, and involves a compensatory response of the pancreatic beta-cell to maintain normoglycemia. However, although pancreatic alpha-cells play a key role in glucose homeostasis and seem to be involved in gestational diabetes, there is no information about their potential adaptations or changes during pregnancy.MATERIAL AND METHODS:Non-pregnant (controls) and pregnant C57BL/6 mice at gestational day 18.5 (G18.5) and their isolated pancreatic islets were used for in vivo and ex vivo studies, respectively. The effect of pregnancy hormones was tested in glucagon-secreting α-TC1.9 cells. Immunohistochemical analysis was performed in pancreatic slices. Glucagon gene expression was monitored by RT-qPCR. Glucagon secretion and plasma hormones were measured by ELISA.RESULTS:Pregnant mice on G18.5 exhibited alpha-cell hypertrophy as well as augmented alpha-cell area and mass. This alpha-cell mass expansion was mainly due to increased proliferation. No changes in alpha-cell apoptosis, ductal neogenesis, or alpha-to-beta transdifferentiation were found compared with controls. Pregnant mice on G18.5 exhibited hypoglucagonemia. Additionally, in vitro glucagon secretion at low glucose levels was decreased in isolated islets from pregnant animals. Glucagon content was also reduced. Experiments in α-TC1.9 cells indicated that, unlike estradiol and progesterone, placental lactogens and prolactin stimulated alpha-cell proliferation. Placental lactogens, prolactin and estradiol also inhibited glucagon release from α-TC1.9 cells at low glucose levels.CONCLUSIONS:The pancreatic alpha-cell in mice undergoes several morphofunctional changes during late pregnancy, which may contribute to proper glucose homeostasis. Gestational hormones are likely involved in these processes.

Many dairy products found in plastics, cosmetics and food containers disrupt human health due to their actions as endocrine-disrupting chemicals (EDCs). Studies in different cellular and animal models have reported that low doses of the EDC bisphenol A (BPA), modify pancreatic β-cell function, induce insulin resistance and other metabolic alterations. Here, we aimed to investigate whether administration of low doses of BPA could regulate ion channels expression and function in mice pancreatic islets. For this purpose, we used microarrays to analyze the global gene expression profile, focusing on the modulation of ion channels-related genes. For in vivo experiments, mice were subcutaneously injected with BPA (100 μg/kg/day during 4 days). Islets were isolated 12 h after the last injection and prepared for RNA extraction and hybridization on Affymetrix GeneChip Mouse Genome 430 2.0 Array. Array scanning and data analysis were performed using Affymetrix Expression Console and the Affymetrix Transcriptome Analysis Console (TAC) Softwares. Expression level analysis was performed using the normalization method based on the processing algorithm called robust multi-array average (RMA). Whole islets or dispersed islets from C57BL/6 or estrogen receptor beta knockout (βERKO) mice were treated in vitro with BPA or vehicle during 48 h. Whole-cell patch-clamp recordings were used to measure sodium and potassium currents. Microarray analysis of islets isolated from mice treated with BPA showed that some ion channels, such as sodium channel voltage-gated type IX, alpha (Scn9a), potassium large conductance calcium-activated channel alpha member 1 (Kcnma1), and Kv channel-interacting protein 1 (Kcnip1), were modulated. These results were confirmed by quantitative RT-PCR. Electrophysiological measurements showed a decrease in both sodium currents and total potassium currents. Additionally, the currents through the voltage-gated potassium channel subunits Kv2.1 and 2.2 as well as the KCa1.1 (encoded by Kcnma1 gene) channels were also reduced. Interestingly, beta-cells from βERKO mice did not present such reductions, suggesting that these effects occur mainly via ERβ. Our results show that, acting as a xenoestrogen, BPA modulates sodium and potassium currents as well as gene expression via ERβ. Funding: Ministerio de Economia y Competitividad, Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), EU Grants SAF2014-58335-P, BFU2017-86579-R, BFU2016-77125-R and Generalitat Valenciana PROMETEO II/2015/016. 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).

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. 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. 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. 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.