Physiology of pancreatic ß-cells: Ion channels and molecular mechanisms implicated in stimulus-secretion coupling.

The human and mouse islet of Langerhans is an endocrine organ composed of five different cells types; insulin-secreting ß-cells, glucagon-producing α-cells, somatostatin-producing δ-cells, pancreatic polypeptide-secreting PP cells and ɛ-cells that secretes ghrelin. The most important cells are the pancreatic ß-cells that comprise around 45-50% of human islets and 75-80% in the mouse. Pancreatic ß-cells secrete insulin at high glucose concentration, thereby finely regulating glycaemia by the hypoglycaemic effects of this hormone. Different ion channels are implicated in the stimulus-secretion coupling of insulin. An increase in the intracellular ATP concentration leads to closure KATP channels, depolarizing the cell and opening voltage-gated calcium channels. The increase of intracellular calcium concentration induced by calcium entry through voltage-gated calcium channels promotes insulin secretion. Here, we briefly describe the diversity of ion channels present in pancreatic ß-cells and the different mechanisms that are responsible to induce insulin secretion in human and mouse cells. Moreover, we described the pathophysiology due to alterations in the physiology of the main ion channels present in pancreatic ß-cell and its implication to predispose metabolic disorders as type 2 diabetes mellitus.

Bisphenol-S and Bisphenol-F alter mouse pancreatic ß-cell ion channel expression and activity and insulin release through an estrogen receptor ERß mediated pathway.

Bisphenol-S (BPS) and Bisphenol-F (BPF) are current Bisphenol-A (BPA) substitutes. Here we used pancreatic ß-cells from wild type (WT) and estrogen receptor ß (ERß) knockout (BERKO) mice to investigate the effects of BPS and BPF on insulin secretion, and the expression and activity of ion channels involved in ß-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 ß-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 ERß pathways. Molecular dynamics simulations indicated differences in ERß ligand-binding domain dimer stabilization and solvation free energy among different bisphenols and PaPE-1. Our data suggest a mode of action involving ERß whose activation alters three key cellular events in ß-cell, namely ion channel expression and activity, and insulin release. These results may help to improve the hazard identification of bisphenols.

Bisphenol A Regulates Sodium Ramp Currents in Mouse Dorsal Root Ganglion Neurons and Increases Nociception.

17ß-Estradiol mediates the sensitivity to pain and is involved in sex differences in nociception. The widespread environmental disrupting chemical bisphenol A (BPA) has estrogenic activity, but its implications in pain are mostly unknown. Here we show that treatment of male mice with BPA (50 µg/kg/day) during 8 days, decreases the latency to pain behavior in response to heat, suggesting increased pain sensitivity. We demonstrate that incubation of dissociated dorsal root ganglia (DRG) nociceptors with 1 nM BPA increases the frequency of action potential firing. SCN9A encodes the voltage-gated sodium channel Nav1.7, which is present in DRG nociceptors and is essential in pain signaling. Nav1.7 and other voltage-gated sodium channels in mouse DRG are considered threshold channels because they produce ramp currents, amplifying small depolarizations and enhancing electrical activity. BPA increased Nav-mediated ramp currents elicited with slow depolarizations. Experiments using pharmacological tools as well as DRG from ERß-/- mice indicate that this BPA effect involves ERα and phosphoinositide 3-kinase. The mRNA expression and biophysical properties other than ramp currents of Nav channels, were unchanged by BPA. Our data suggest that BPA at environmentally relevant doses affects the ability to detect noxious stimuli and therefore should be considered when studying the etiology of pain conditions.

Oestrogen receptor ß 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 ß (ERß). 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 ß (ERß) knockout (Erß-/-) 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 ERß-/- mice did not present BPA-induced changes, suggesting that ERß 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 ERß 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.

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 ERß 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, ERß, 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 bile acid TUDCA increases glucose-induced insulin secretion via the cAMP/PKA pathway in pancreatic beta cells.

OBJECTIVE: While bile acids are important for the digestion process, they also act as signaling molecules in many tissues, including the endocrine pancreas, which expresses specific bile acid receptors that regulate several cell functions. In this study, we investigated the effects of the conjugated bile acid TUDCA on glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells. METHODS: Pancreatic islets were isolated from 90-day-old male mice. Insulin secretion was measured by radioimmunoassay, protein phosphorylation by western blot, Ca(2+) signals by fluorescence microscopy and ATP-dependent K(+) (KATP) channels by electrophysiology. RESULTS: TUDCA dose-dependently increased GSIS in fresh islets at stimulatory glucose concentrations but remained without effect at low glucose levels. This effect was not associated with changes in glucose metabolism, Ca(2+) signals or KATP channel activity; however, it was lost in the presence of a cAMP competitor or a PKA inhibitor. Additionally, PKA and CREB phosphorylation were observed after 1-hour incubation with TUDCA. The potentiation of GSIS was blunted by the Gα stimulatory, G protein subunit-specific inhibitor NF449 and mimicked by the specific TGR5 agonist INT-777, pointing to the involvement of the bile acid G protein-coupled receptor TGR5. CONCLUSION: Our data indicate that TUDCA potentiates GSIS through the cAMP/PKA pathway.

Antidiabetic actions of an estrogen receptor ß selective agonist.

The estrogen receptor ß (ERß) is emerging as an important player in the physiology of the endocrine pancreas. We evaluated the role and antidiabetic actions of the ERß selective agonist WAY200070 as an insulinotropic molecule. We demonstrate that WAY200070 enhances glucose-stimulated insulin secretion both in mouse and human islets. In vivo experiments showed that a single administration of WAY200070 leads to an increase in plasma insulin levels with a concomitant improved response to a glucose load. Two-week treatment administration increased glucose-induced insulin release and pancreatic ß-cell mass and improved glucose and insulin sensitivity. In addition, streptozotocin-nicotinamide-induced diabetic mice treated with WAY200070 exhibited a significant improvement in plasma insulin levels and glucose tolerance as well as a regeneration of pancreatic ß-cell mass. Studies performed in db/db mice demonstrated that this compound restored first-phase insulin secretion and enhanced pancreatic ß-cell mass. We conclude that ERß agonists should be considered as new targets for the treatment of diabetes.

Insulinotropic effect of the non-steroidal compound STX in pancreatic ß-cells.

The non-steroidal compound STX modulates the hypothalamic control of core body temperature and energy homeostasis. The aim of this work was to study the potential effects of STX on pancreatic ß-cell function. 1-10 nM STX produced an increase in glucose-induced insulin secretion in isolated islets from male mice, whereas it had no effect in islets from female mice. This insulinotropic effect of STX was abolished by the anti-estrogen ICI 182,780. STX increased intracellular calcium entry in both whole islets and isolated ß-cells, and closed the K(ATP) channel, suggesting a direct effect on ß-cells. When intraperitoneal glucose tolerance test was performed, a single dose of 100 µg/kg body weight STX improved glucose sensitivity in males, yet it had a slight effect on females. In agreement with the effect on isolated islets, 100 µg/kg dose of STX enhanced the plasma insulin increase in response to a glucose load, while it did not in females. Long-term treatment (100 µg/kg, 6 days) of male mice with STX did not alter body weight, fasting glucose, glucose sensitivity or islet insulin content. Ovariectomized females were insensitive to STX (100 µg/kg), after either an acute administration or a 6-day treatment. This long-term treatment was also ineffective in a mouse model of mild diabetes. Therefore, STX appears to have a gender-specific effect on blood glucose homeostasis, which is only manifested after an acute administration. The insulinotropic effect of STX in pancreatic ß-cells is mediated by the closure of the K(ATP) channel and the increase in intracellular calcium concentration. The in vivo improvement in glucose tolerance appears to be mostly due to the enhancement of insulin secretion from ß-cells.

Rapid insulinotropic action of low doses of bisphenol-A on mouse and human islets of Langerhans: role of estrogen receptor ß.

Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical (EDC) used as the base compound in the manufacture of polycarbonate plastics. It alters pancreatic ß-cell function and can be considered a risk factor for type 2 diabetes in rodents. Here we used ERß-/- mice to study whether ERß is involved in the rapid regulation of K(ATP) channel activity, calcium signals and insulin release elicited by environmentally relevant doses of BPA (1 nM). We also investigated these effects of BPA in ß-cells and whole islets of Langerhans from humans. 1 nM BPA rapidly decreased K(ATP) channel activity, increased glucose-induced [Ca(2+)](i) signals and insulin release in ß-cells from WT mice but not in cells from ERß-/- mice. The rapid reduction in the K(ATP) channel activity and the insulinotropic effect was seen in human cells and islets. BPA actions were stronger in human islets compared to mouse islets when the same BPA concentration was used. Our findings suggest that BPA behaves as a strong estrogen via nuclear ERß and indicate that results obtained with BPA in mouse ß-cells may be extrapolated to humans. This supports that BPA should be considered as a risk factor for metabolic disorders in humans.

Bisphenol-A acts as a potent estrogen via non-classical estrogen triggered pathways.

Bisphenol-A (BPA) is an estrogenic monomer commonly used in the manufacture of numerous consumer products such as food and beverage containers. Widespread human exposure to significant doses of this compound has been reported. Traditionally, BPA has been considered a weak estrogen, based on its lower binding affinity to the nuclear estrogen receptors (ERs) compared to 17-ß estradiol (E2) as well as its low transcriptional activity after ERs activation. However, in vivo animal studies have demonstrated that it can interfere with endocrine signaling pathways at low doses during fetal, neonatal or perinatal periods as well as in adulthood. In addition, mounting evidence suggests a variety of pathways through which BPA can elicit cellular responses at very low concentrations with the same or even higher efficiency than E2. Thus, the purpose of the present review is to analyze with substantiated scientific evidence the strong estrogenic activity of BPA when it acts through alternative mechanisms of action at least in certain cell types.

Involvement of ATP-sensitive potassium (K(ATP)) channels in the loss of beta-cell function induced by human islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is a major component of amyloid deposition in pancreatic islets of patients with type 2 diabetes. It is known that IAPP can inhibit glucose-stimulated insulin secretion; however, the mechanisms of action have not yet been established. In the present work, using a rat pancreatic beta-cell line, INS1E, we have created an in vitro model that stably expressed human IAPP gene (hIAPP cells). These cells showed intracellular oligomers and a strong alteration of glucose-stimulated insulin and IAPP secretion. Taking advantage of this model, we investigated the mechanism by which IAPP altered beta-cell secretory response and contributed to the development of type 2 diabetes. We have measured the intracellular Ca(2+) mobilization in response to different secretagogues as well as mitochondrial metabolism. The study of calcium signals in hIAPP cells demonstrated an absence of response to glucose and also to tolbutamide, indicating a defect in ATP-sensitive potassium (K(ATP)) channels. Interestingly, hIAPP showed a greater maximal respiratory capacity than control cells. These data were confirmed by an increased mitochondrial membrane potential in hIAPP cells under glucose stimulation, leading to an elevated reactive oxygen species level as compared with control cells. We concluded that the hIAPP overexpression inhibits insulin and IAPP secretion in response to glucose affecting the activity of K(ATP) channels and that the increased mitochondrial metabolism is a compensatory response to counteract the secretory defect of beta-cells.

Regulation of K(ATP) channel by 17ß-estradiol in pancreatic ß-cells.

ATP-sensitive potassium channels (K(ATP)) regulate electrical activity and insulin secretion in pancreatic ß-cells. When glucose concentration increases, the [ATP]/[ADP] ratio rises closing K(ATP) channels, and the membrane potential depolarizes, triggering insulin secretion. This pivotal role of K(ATP) channels is used not only by glucose but also by neurotransmitters, hormones and other physiological agents to modulate electrical and secretory ß-cell response. In recent years, it has been demonstrated that estrogens and estrogen receptors are involved in glucose homeostasis, and that they can modulate the electrical activity and insulin secretion of pancreatic ß-cells. The hormone 17ß-estradiol (E2), at physiological levels, is implicated in maintaining normal insulin sensitivity for ß-cell function. Long term exposure to E2 increases insulin content, insulin gene expression and insulin release via the estrogen receptor α (ERα), while rapid responses to E2 can regulate K(ATP) channels increasing cGMP levels through the estrogen receptor ß (ERß) and type A guanylate cyclase receptor (GC-A). This review summarizes the main actions of 17ß-estradiol on K(ATP) channels and the subsequent insulin release in pancreatic ß-cells.

Role of estrogen receptors alpha, beta and GPER1/GPR30 in pancreatic beta-cells.

Estrogen receptors (ER) are emerging as important molecules involved in the adaptation of beta-cells to insulin resistance. The onset of type 2 diabetes is marked by insulin secretory dysfunction and decreased beta-cell mass. During pregnancy, puberty and obesity there is increased metabolic demand and insulin resistance is developed. This metabolic state increases the demand on beta-cells to augment insulin biosynthesis and release. In this respect, ERalpha is directly implicated in the E2-regulation of insulin content and secretion, while ERbeta is in the E2-potentiation of glucose-induced insulin release. Both receptors develop their actions within the physiological range of E2. In addition, the G protein-coupled estrogen receptor (GPER1/GPR30) seems to be implicated in the E2-regulation of stimulus-secretion coupling in the three cell types of the islet. The increased demand of insulin production for long time may lead to beta-cell stress and apoptosis. ERalpha, ERbeta and GPER1/GPR30 are involved in preventing beta-cell apoptosis, impeding the loss of critical beta-cell mass. Therefore, estrogen receptors may play an essential role in the adaptation of the pancreas to insulin resistant periods.

Bisphenol-A: a new diabetogenic factor?

The aim of this review was to analyze the potential effects of environmental chemicals on homeostatic control related to glycemia and energy balance. Many of the environmental chemicals can mimic or interfere with the action of hormones and are generally referred to as "endocrine disruptors". Among these compounds, polychlorinated biphenyls, dioxins, phthalates and bisphenol-A have been correlated with alterations in blood glucose homeostasis in humans. In rodents it has been demonstrated that small doses of bisphenol-A have profound effects on glucose metabolism. Therefore, this altered blood glucose homeostasis may enhance the development of type 2 diabetes.

Rapid regulation of K(ATP) channel activity by 17{beta}-estradiol in pancreatic {beta}-cells involves the estrogen receptor {beta} and the atrial natriuretic peptide receptor.

The ATP-sensitive potassium (K(ATP)) channel is a key molecule involved in glucose-stimulated insulin secretion. The activity of this channel regulates beta-cell membrane potential, glucose- induced [Ca(2+)](i) signals, and insulin release. In this study, the rapid effect of physiological concentrations of 17beta-estradiol (E2) on K(ATP) channel activity was studied in intact beta-cells by use of the patch-clamp technique. When cells from wild-type (WT) mice were used, 1 nm E2 rapidly reduced K(ATP) channel activity by 60%. The action of E2 on K(ATP) channel was not modified in beta-cells from ERalpha-/- mice, yet it was significantly reduced in cells from ERbeta-/- mice. The effect of E2 was mimicked by the ERbeta agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN). Activation of ERbeta by DPN enhanced glucose-induced Ca(2+) signals and insulin release. Previous evidence indicated that the acute inhibitory effects of E2 on K(ATP) channel activity involve cyclic GMP and cyclic GMP-dependent protein kinase. In this study, we used beta-cells from mice with genetic ablation of the membrane guanylate cyclase A receptor for atrial natriuretic peptide (also called the atrial natriuretic peptide receptor) (GC-A KO mice) to demonstrate the involvement of this membrane receptor in the rapid E2 actions triggered in beta-cells. E2 rapidly inhibited K(ATP) channel activity and enhanced insulin release in islets from WT mice but not in islets from GC-A KO mice. In addition, DPN reduced K(ATP) channel activity in beta-cells from WT mice, but not in beta-cells from GC-A KO mice. This work unveils a new role for ERbeta as an insulinotropic molecule that may have important physiological and pharmacological implications.

The role of oestrogens in the adaptation of islets to insulin resistance.

Pregnancy is characterized by peripheral insulin resistance, which is developed in parallel with a plasma increase of maternal hormones; these include prolactin, placental lactogens, progesterone and oestradiol among others. Maternal insulin resistance is counteracted by the adaptation of the islets of Langerhans to the higher insulin demand. If this adjustment is not produced, gestational diabetes may be developed. The adaptation process of islets is characterized by an increase of insulin biosynthesis, an enhanced glucose-stimulated insulin secretion (GSIS) and an increase of beta-cell mass. It is not completely understood why, in some individuals, beta-cell mass and function fail to adapt to the metabolic demands of pregnancy, yet a disruption of the beta-cell response to maternal hormones may play a key part. The role of the maternal hormone 17beta-oestradiol (E2) in this adaptation process has been largely unknown. However, in recent years, it has been demonstrated that E2 acts directly on beta-cells to increase insulin biosynthesis and to enhance GSIS through different molecular mechanisms. E2 does not increase beta-cell proliferation but it is involved in beta-cell survival. Classical oestrogen receptors ERalpha and ERbeta, as well as the G protein-coupled oestrogen receptor (GPER) seem to be involved in these adaptation changes. In addition, as the main production of E2 in post-menopausal women comes from the adipose tissue, E2 may act as a messenger between adipocytes and islets in obesity.

The pancreatic beta-cell as a target of estrogens and xenoestrogens: Implications for blood glucose homeostasis and diabetes.

The estrogen receptor ERalpha is emerging as a key molecule involved in glucose and lipid metabolism. The main functions of pancreatic beta-cells are the biosynthesis and release of insulin, the only hormone that can directly decrease blood glucose levels. Estrogen receptors ERalpha and ERbeta exist in beta-cells. The role of ERbeta is still unknown, yet ERalpha plays an important role in the regulation of insulin biosynthesis, insulin secretion and beta-cell survival. Activation of ERalpha by 17beta-estradiol (E2) and the environmental estrogen bisphenol-A (BPA) promotes an increase of insulin biosynthesis through a non-classical estrogen-activated pathway that involves phosphorylation of ERK1/2. The activation of ERalpha by physiological concentrations of E2 may play an important role in the adaptation of the endocrine pancreas to pregnancy. However, if ERalpha is over stimulated by an excess of E2 or the action of an environmental estrogen such as BPA, it will produce an excessive insulin signaling. This may provoke insulin resistance in the liver and muscle, as well as beta-cell exhaustion and therefore, it may contribute to the development of type II diabetes.