Interactions between Atorvastatin and the Farnesoid X Receptor Impair Insulinotropic Effects of Bile Acids and Modulate Diabetogenic Risk.

Bile acids such as chenodeoxycholic acid (CDC) acutely enhance insulin secretion via the farnesoid X receptor (FXR). Statins, which are frequently prescribed for patients with type 2 diabetes who suffer from dyslipidemia, are known for their diabetogenic risk and are reported to interact with the FXR. Our study investigates whether this interaction is relevant for beta cell signaling and plays a role for negative effects of statins on glycemic control. Experiments were performed with islets and islet cells from C57BL/6N wild-type and FXR-knockout (KO) mice. Culturing islets with atorvastatin (15 µM) for 24 hours decreased glucose-stimulated insulin secretion by approximately 30% without affecting ATP synthesis. Prolonged exposure for 7 days lowered the concentration necessary for impairment of insulin release to 150 nM. After 24-hour culture with atorvastatin, the ability of CDC (500 nM) to elevate [Ca2+]c was diminished and the potentiating effect on insulin secretion was completely lost. Mevalonate largely reduced the negative effect of atorvastatin. Nuclear activity of FXR was reduced by atorvastatin in a mouse FXR reporter assay. The atorvastatin-induced decrease in insulin release was also present in FXR-KO mice. Although not a prerequisite, FXR seems to influence the degree of damage caused by atorvastatin depending on its interaction with CDC: Preparations responding to CDC with an increase in insulin secretion under control conditions were less impaired by atorvastatin than preparations that were nonresponsive to CDC. Extended stimulation of FXR by the synthetic agonist GW4064, which is suggested to induce translocation of FXR from the cytosol into the nucleus, increased the inhibitory effect of atorvastatin. In conclusion, atorvastatin inhibits insulin release and prevents positive effects of bile acids on beta cell function. Both interactions may contribute to progression of type 2 diabetes mellitus. SIGNIFICANCE STATEMENT: This study shows that the diabetogenic risk of statins is coupled to the activity of farnesoid X receptor (FXR)-dependent signaling pathways in beta cells. On the one hand, statins abolish the insulinotropic effects of bile acids and on the other hand, FXR determines the level of impairment of islet function by the statin.

ATP binding without hydrolysis switches sulfonylurea receptor 1 (SUR1) to outward-facing conformations that activate KATP channels.

Neuroendocrine-type ATP-sensitive K+ (KATP) channels are metabolite sensors coupling membrane potential with metabolism, thereby linking insulin secretion to plasma glucose levels. They are octameric complexes, (SUR1/Kir6.2)4, comprising sulfonylurea receptor 1 (SUR1 or ABCC8) and a K+-selective inward rectifier (Kir6.2 or KCNJ11). Interactions between nucleotide-, agonist-, and antagonist-binding sites affect channel activity allosterically. Although it is hypothesized that opening these channels requires SUR1-mediated MgATP hydrolysis, we show here that ATP binding to SUR1, without hydrolysis, opens channels when nucleotide antagonism on Kir6.2 is minimized and SUR1 mutants with increased ATP affinities are used. We found that ATP binding is sufficient to switch SUR1 alone between inward- or outward-facing conformations with low or high dissociation constant, KD , values for the conformation-sensitive channel antagonist [3H]glibenclamide ([3H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's KD for [3H]GBM. This reduction required the Kir N terminus (KNtp), consistent with KNtp occupying a "transport cavity," thus positioning it to link ATP-induced SUR1 conformational changes to channel gating. Moreover, ATP/GBM site coupling was constrained in WT SUR1/WT Kir6.2 channels; ATP-bound channels had a lower KD for [3H]GBM than ATP-bound SUR1. This constraint was largely eliminated by the Q1179R neonatal diabetes-associated mutation in helix 15, suggesting that a "swapped" helix pair, 15 and 16, is part of a structural pathway connecting the ATP/GBM sites. Our results suggest that ATP binding to SUR1 biases KATP channels toward open states, consistent with SUR1 variants with lower KD values causing neonatal diabetes, whereas increased KD values cause congenital hyperinsulinism.

Energy depletion and not ROS formation is a crucial step of glucolipotoxicity (GLTx) in pancreatic beta cells.

We have shown previously that genetic or pharmacological deletion of KATP channels protect against beta cell dysfunction induced by reactive oxygen species (ROS). Since it is assumed that glucolipotoxicity (GLTx) causes ROS production, we aimed to evaluate whether suppression of KATP channel activity can also prevent beta cell damage evoked by GLTx. We used an in vitro model of GLTx and measured distinct parameters of stimulus-secretion coupling. GLTx gradually induced disturbances of Ca2+ oscillations over 3 days. This impairment in Ca2+ dynamics was partially reversed in beta cells without functional KATP channels (SUR1-/-) and by the sulfonylurea gliclazide but not by tolbutamide. By contrast, the GLTx-induced suppression of glucose-induced insulin secretion could not be rescued by decreased KATP channel activity pointing to a direct interaction of GLTx with the secretory capacity. Accordingly, GLTx also suppressed KCl-induced insulin secretion. GLTx was not accompanied by decisively increased ROS production or enhanced apoptosis. Insulin content of beta cells was markedly reduced by GLTx, an effect not prevented by gliclazide. Since GLTx markedly diminished the mitochondrial membrane potential and cellular ATP content, lack of ATP is assumed to decrease insulin biosynthesis. The deleterious effect of GLTx is therefore caused by direct interference with the secretory capacity whereby reduction of insulin content is one important parameter. These findings deepen our understanding how GLTx damages beta cells and reveal that GLTx is disconnected from ROS formation, a notion important for targeting beta cells in the treatment of diabetes. Overall, GLTx-induced energy depletion may be a primary step in the cascade of events leading to loss of beta cell function in type-2 diabetes mellitus.

Mitochondrial succinate dehydrogenase is involved in stimulus-secretion coupling and endogenous ROS formation in murine beta cells.

AIMS/HYPOTHESIS: Generation of reduction equivalents is a prerequisite for nutrient-stimulated insulin secretion. Mitochondrial succinate dehydrogenase (SDH) fulfils a dual function with respect to mitochondrial energy supply: (1) the enzyme is part of mitochondrial respiratory chains; and (2) it catalyses oxidation of succinate to fumarate in the Krebs cycle. The aim of our study was to elucidate the significance of SDH for beta cell stimulus-secretion coupling (SSC). METHODS: Mitochondrial variables, reactive oxygen species (ROS) and cytosolic Ca(2+) concentration ([Ca(2+)]c) were measured by fluorescence techniques and insulin release by radioimmunoassay in islets or islet cells of C57Bl/6N mice. RESULTS: Inhibition of SDH with 3-nitropropionic acid (3-NPA) or monoethyl fumarate (MEF) reduced glucose-stimulated insulin secretion. Inhibition of the ATP-sensitive K(+) channel (KATP channel) partly prevented this effect, whereas potentiation of antioxidant defence by superoxide dismutase mimetics (TEMPOL and mito-TEMPO) or by nuclear factor erythroid 2-related factor 2 (Nrf-2)-mediated upregulation of antioxidant enzymes (oltipraz, tert-butylhydroxyquinone) did not diminish the inhibitory influence of 3-NPA. Blocking SDH decreased glucose-stimulated increase in intracellular FADH2 concentration without alterations in NAD(P)H. In addition, 3-NPA and MEF drastically reduced glucose-induced hyperpolarisation of mitochondrial membrane potential, indicative of decreased ATP production. As a consequence, the glucose-stimulated rise in [Ca(2+)]c was significantly delayed and reduced. Acute application of 3-NPA interrupted glucose-driven oscillations of [Ca(2+)]c. 3-NPA per se did not elevate intracellular ROS, but instead prevented glucose-induced ROS accumulation. CONCLUSIONS/INTERPRETATION: SDH is an important regulator of insulin secretion and ROS production. Inhibition of SDH interrupts membrane-potential-dependent SSC, pointing to a pivotal role of mitochondrial FAD/FADH2 homeostasis for the maintenance of glycaemic control.

Evidence against a Ca(2+)-induced potentiation of dehydrogenase activity in pancreatic beta-cells.

Pancreatic beta-cells respond to an unchanging stimulatory glucose concentration with oscillations in membrane potential (Vm), cytosolic Ca(2+) concentration ([Ca(2+)]c), and insulin secretion. The underlying mechanisms are largely ascertained. Some particular details, however, are still in debate. Stimulus-secretion coupling (SSC) of beta-cells comprises glucose-induced Ca(2+) influx into the cytosol and thus into mitochondria. It is suggested that this activates (mitochondrial) dehydrogenases leading to an increase in reduction equivalents and ATP production. According to SSC, a glucose-induced increase in ATP production would thus further augment ATP production, i.e. induce a feed-forward loop that is hardly compatible with oscillations. Consistently, other studies favour a feedback mechanism that drives oscillatory mitochondrial ATP production. If Ca(2+) influx activates dehydrogenases, a change in [Ca(2+)]c should increase the concentration of reduction equivalents. We measured changes in flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) autofluorescence in response to changes in glucose concentration or glucose-independent changes in [Ca(2+)]c. The FAD signal was altered by glucose but not by alterations in [Ca(2+)]c. NAD(P)H was increased by glucose but even decreased by Ca(2+) influx evoked by tolbutamide. The mitochondrial membrane potential ΔΨ was hyperpolarized by 4 mM glucose. As adding tolbutamide then depolarized ΔΨ, we deduce that Ca(2+) does not activate mitochondrial activity but by contrast even inhibits it by reducing the driving force for ATP production. Inhibition of Ca(2+) influx reversed the Ca(2+)-induced changes in ΔΨ and NAD(P)H. The results are consistent with a feedback mechanism which transiently and repeatedly reduces ATP production and explain the oscillatory activity of pancreatic beta-cells at increased glucose concentrations.

Glitazones exert multiple effects on ß-cell stimulus-secretion coupling.

Earlier studies suggest that glitazones exert beneficial effects in patients with type 2 diabetes by directly affecting insulin secretion of ß-cells, besides improving the effectiveness of insulin in peripheral tissues. The effects of glitazones on stimulus-secretion coupling (SSC) are poorly understood. We tested the influence of troglitazone and pioglitazone on different parameters of SSC, including insulin secretion (radioimmunoassay), cell membrane potential, various ion currents (patch-clamp), mitochondrial membrane potential (ΔΨ), and cytosolic Ca(2+) concentration (fluorescence). Troglitazone exerted stimulatory, inhibitory, or no effects on insulin secretion depending on the drug and glucose concentration. It depolarized the ΔΨ, thus lowering ATP production, which resulted in opening of ATP-dependent K(+) channels (K(ATP) channels) and reduced insulin secretion. However, it also exerted direct inhibitory effects on K(ATP) channels that can explain enhanced insulin secretion. Troglitazone also inhibited the currents through voltage-dependent Ca(2+) and K(+) channels. Pioglitazone was less effective than troglitazone on all parameters tested. The effects of both glitazones were markedly reduced in the presence of bovine serum albumin. Glitazones exert multiple actions on ß-cell SSC that have to be considered as undesired side effects because the influence of these compounds on ß-cells is not controllable. The final effect on insulin secretion depends on many parameters, including the actual glucose and drug concentration, protein binding of the drug, and the drug by itself. Troglitazone and pioglitazone differ in their influence on SSC. It can be assumed that the effects of pioglitazone on ß-cells are negligible under in vivo conditions.

Rapid functional evaluation of beta-cells by extracellular recording of membrane potential oscillations with microelectrode arrays.

The membrane potential (V (m)) of beta-cells oscillates at glucose concentrations between ~6 and 25 mM, i.e. burst phases with action potentials alternate with silent interburst phases generating so-called slow waves. The slow waves drive oscillations of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) and insulin secretion. The length of the bursts correlates with the amount of insulin release. Thus, the fraction of plateau phase (FOPP), i.e. the percentage of time with burst activity, is an excellent marker for beta-cell function and metabolic integrity. Extracellular voltage changes of mouse islets were measured using a microelectrode array (MEA) allowing the detection of burst and interburst phases. At a non-stimulating glucose concentration (3 mM) no electrical activity was detectable while bursting was continuous at 30 mM. The glucose concentration-response (determined as FOPP) curve revealed half-maximal stimulation at 12 ± 1 mM (Hill equation fit). The signal was sensitive to K(ATP) channel modulators, e.g. tolbutamide or diazoxide. Simultaneous recordings of electrical activity and [Ca(2+)](c) revealed congruent bursts and peaks, respectively. The extracellular recordings are in perfect agreement with more time-consuming intracellular electrical recordings. The results provide a 'proof-of-principle' for detection of beta-cell slow waves and determination of the FOPP using extracellular electrodes in a MEA-based system. The method is facile and provides the capability to study the effects of modulators of beta-cell function including possible anti-diabetic drugs in real time. Moreover, the method may be useful for checking the metabolic integrity of human donor islets prior to transplantation.

Acyl-Ghrelin Influences Pancreatic ß-Cell Function by Interference with KATP Channels.

The aim for this study was to elucidate how the hypothalamic hunger-inducing hormone acyl-ghrelin (AG), which is also produced in the pancreas, affects ß-cell function, with particular attention to the role of ATP-sensitive K+ (KATP) channels and the exact site of action of the hormone. AG hyperpolarized the membrane potential and decreased cytoplasmic calcium concentration [Ca2+]c and glucose-stimulated insulin secretion (GSIS). These effects were abolished in ß-cells from SUR1-knockout (KO) mice. AG increased KATP current but only in a configuration with intact metabolism. Unacylated ghrelin counteracted the effects of AG. The influence of AG on membrane potential and GSIS could only be averted in the combined presence of a ghrelin receptor (GHSR1a) antagonist and an inverse agonist. The inhibition of GSIS by AG could be prevented by dibutyryl cyclic-cAMP or 3-isobutyl-1-methylxanthine and the somatostatin (SST) receptor 2-5 antagonist H6056. These data indicate that AG indirectly opens KATP channels probably by interference with the cAMP/cAMP-dependent protein kinase pathway, resulting in a decrease of [Ca2+]c and GSIS. The experiments with SUR1-KO ß-cells point to a direct effect of AG on ß-cells and not, as earlier suggested, to an exclusive effect by AG-induced SST release from δ-cells. Nevertheless, SST receptors may be involved in the effect of AG, possibly by heteromerization of AG and SST receptors.

Oxidative stress and beta-cell dysfunction.

Diabetes mellitus type 1 and 2 (T1DM and T2DM) are complex multifactorial diseases. Loss of beta-cell function caused by reduced secretory capacity and enhanced apoptosis is a key event in the pathogenesis of both diabetes types. Oxidative stress induced by reactive oxygen and nitrogen species is critically involved in the impairment of beta-cell function during the development of diabetes. Because of their low antioxidant capacity, beta-cells are extremely sensitive towards oxidative stress. In beta-cells, important targets for an oxidant insult are cell metabolism and K(ATP) channels. The oxidant-evoked alterations of K(ATP) channel activity seem to be critical for oxidant-induced dysfunction because genetic ablation of K(ATP) channels attenuates the effects of oxidative stress on beta-cell function. Besides the effects on metabolism, interference of oxidants with mitochondria induces key events in apoptosis. Consequently, increasing antioxidant defence is a promising strategy to delay beta cell failure in (pre)-diabetic patients or during islet transplantation. Knock-out of K(ATP) channels has beneficial effects on oxidant-induced inhibition of insulin secretion and cell death. Interestingly, these effects can be mimicked by sulfonylureas that have been used in the treatment of T2DM for many years. Loss of functional K(ATP) channels leads to up-regulation of antioxidant enzymes, a process that depends on cytosolic Ca(2+). These observations are of great importance for clinical intervention because they show a possibility to protect beta-cells at an early stage before dramatic changes of the secretory capacity and loss of cell mass become manifest and lead to glucose intolerance or even overt diabetes.

Electrophysiology of islet cells.

Stimulus-Secretion Coupling (SSC) of pancreatic islet cells comprises electrical activity. Changes of the membrane potential (V(m)) are regulated by metabolism-dependent alterations in ion channel activity. This coupling is best explored in beta-cells. The effect of glucose is directly linked to mitochondrial metabolism as the ATP/ADP ratio determines the open probability of ATP-sensitive K(+) channels (K(ATP) channels). Nucleotide sensitivity and concentration in the direct vicinity of the channels are controlled by several factors including phospholipids, fatty acids, and kinases, e.g., creatine and adenylate kinase. Closure of K(ATP) channels leads to depolarization of beta-cells via a yet unknown depolarizing current. Ca(2+) influx during action potentials (APs) results in an increase of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that triggers exocytosis. APs are elicited by the opening of voltage-dependent Na(+) and/or Ca(2+) channels and repolarized by voltage- and/or Ca(2+)-dependent K(+) channels. At a constant stimulatory glucose concentration APs are clustered in bursts that are interrupted by hyperpolarized interburst phases. Bursting electrical activity induces parallel fluctuations in [Ca(2+)](c) and insulin secretion. Bursts are terminated by I(Kslow) consisting of currents through Ca(2+)-dependent K(+) channels and K(ATP) channels. This review focuses on structure, characteristics, physiological function, and regulation of ion channels in beta-cells. Information about pharmacological drugs acting on K(ATP) channels, K(ATP) channelopathies, and influence of oxidative stress on K(ATP) channel function is provided. One focus is the outstanding significance of L-type Ca(2+) channels for insulin secretion. The role of less well characterized beta-cell channels including voltage-dependent Na(+) channels, volume sensitive anion channels (VSACs), transient receptor potential (TRP)-related channels, and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is discussed. A model of beta-cell oscillations provides insight in the interplay of the different channels to induce and maintain electrical activity. Regulation of beta-cell electrical activity by hormones and the autonomous nervous system is discussed. alpha- and delta-cells are also equipped with K(ATP) channels, voltage-dependent Na(+), K(+), and Ca(2+) channels. Yet the SSC of these cells is less clear and is not necessarily dependent on K(ATP) channel closure. Different ion channels of alpha- and delta-cells are introduced and SSC in alpha-cells is described in special respect of paracrine effects of insulin and GABA secreted from beta-cells.

Approved LXR agonists exert unspecific effects on pancreatic ß-cell function.

Novel agonists of the nuclear liver-X-receptor (LXR) are designed to treat metabolic disorders or cancer. The rationale to develop these new drugs is based on promising results with established LXR agonist like T0901317 and GW3965. LXRα and LXRß are expressed in ß-cells, and expression is increased by T0901317. The aim of the present study was to evaluate whether effects of these drugs on ß-cell function are specific and reliably linked to LXR activation. T0901317 and GW3965, widely used as specific LXR agonists, show rapid, non-genomic effects on stimulus-secretion coupling of mouse pancreatic ß-cells at low µM concentrations. T0901317 lowered the cytosolic Ca2+ concentration, reduced or completely inhibited action potentials, and decreased insulin secretion. GW3965 exerted similar effects on insulin secretion. T0901317 affected the production of reactive oxygen species and ATP. The involvement of the classical nuclear LXRs in T0901317- and GW3965-mediated effects in ß-cells could be ruled out using LXRα, LXRß and double knockout mice. Our results strongly suggest that LXR agonists, that are considered to be specific for this receptor, interfere with mitochondrial metabolism and metabolism-independent processes in ß-cells. Thus, it is indispensable to test novel LXR agonists accompanying to ongoing clinical trials for acute and chronic effects on cell function in cellular systems and/or animal models lacking classical LXRs.

Possible New Strategies for the Treatment of Congenital Hyperinsulinism.

Objective: Congenital hyperinsulinism (CHI) is a rare disease characterized by persistent hypoglycemia as a result of inappropriate insulin secretion, which can lead to irreversible neurological defects in infants. Poor efficacy and strong adverse effects of the current medications impede successful treatment. The aim of the study was to investigate new approaches to silence ß-cells and thus attenuate insulin secretion. Research Design and Methods: In the scope of our research, we tested substances more selective and more potent than the gold standard diazoxide that also interact with neuroendocrine ATP-sensitive K+ (KATP) channels. Additionally, KATP channel-independent targets as Ca2+-activated K+ channels of intermediate conductance (KCa3.1) and L-type Ca2+ channels were investigated. Experiments were performed using human islet cell clusters isolated from tissue of CHI patients (histologically classified as pathological) and islet cell clusters obtained from C57BL/6N (WT) or SUR1 knockout (SUR1-/-) mice. The cytosolic Ca2+ concentration ([Ca2+]c) was used as a parameter for the pathway regulated by electrical activity and was determined by fura-2 fluorescence. The mitochondrial membrane potential (ΔΨ) was determined by rhodamine 123 fluorescence and single channel currents were measured by the patch-clamp technique. Results: The selective KATP channel opener NN414 (5 µM) diminished [Ca2+]c in isolated human CHI islet cell clusters and WT mouse islet cell clusters stimulated with 10 mM glucose. In islet cell clusters lacking functional KATP channels (SUR1-/-) the drug was without effect. VU0071063 (30 µM), another KATP channel opener considered to be selective, lowered [Ca2+]c in human CHI islet cell clusters. The compound was also effective in islet cell clusters from SUR1-/- mice, showing that [Ca2+]c is influenced by additional effects besides KATP channels. Contrasting to NN414, the drug depolarized ΔΨ in murine islet cell clusters pointing to severe interference with mitochondrial metabolism. An opener of KCa3.1 channels, DCEBIO (100 µM), significantly decreased [Ca2+]c in SUR1-/- and human CHI islet cell clusters. To target L-type Ca2+ channels we tested two already approved drugs, dextromethorphan (DXM) and simvastatin. DXM (100 µM) efficiently diminished [Ca2+]c in stimulated human CHI islet cell clusters as well as in stimulated SUR1-/- islet cell clusters. Similar effects on [Ca2+]c were observed in experiments with simvastatin (7.2 µM). Conclusions: NN414 seems to provide a good alternative to the currently used KATP channel opener diazoxide. Targeting KCa3.1 channels by channel openers or L-type Ca2+ channels by DXM or simvastatin might be valuable approaches for treatment of CHI caused by mutations of KATP channels not sensitive to KATP channel openers.

Glucose Responsiveness of ß-Cells Depends on Fatty Acids.

Glucose-stimulated insulin secretion (GSIS) is the gold standard for ß-cell function. Both experimental and clinical diabetology, i. e., preceding transplantation of isolated human islets, depend on functional testing. However, multiple factors influence GSIS rendering the comparison of different in vitro tests of glucose responsiveness difficult. This study examined the influence of bovine serum albumin (BSA)-coupled fatty acids on GSIS. Isolated islet preparations of human donors and of 12-months old mice displayed impaired GSIS in the presence of 0.5% FFA-free BSA compared to 0.5% BSA (fraction V, not deprived from fatty acids). In aged INS-1E cells, i. e. at a high passage number, GSIS became highly sensitive to FFA-free BSA. Readdition of 30 µM palmitate or 30 µM oleate to FFA-free BSA did not rescue GSIS, while the addition of 100 µM palmitate and the raise of extracellular Ca2+from 1.3 to 2.6 mM improved glucose responsiveness. A high concentration of palmitate (600 µM), which fully activates FFA1, largely restored insulin secretion. The FFA1-agonist TUG-469 also increased insulin secretion but to a lesser extent than palmitate. Glucose- and TUG-induced Ca2+oscillations were impaired in glucose-unresponsive, i. e., aged INS-1E cells. These results suggest that fatty acid deprivation (FFA-free BSA) impairs GSIS mainly through an effect on Ca2+sensitivity.

PET/MRI enables simultaneous in vivo quantification of ß-cell mass and function.

Non-invasive imaging of ß-cells represents a desirable preclinical and clinical tool to monitor the change of ß-cell mass and the loss of function during pre-diabetic stages. Although it is widely accepted that manganese (Mn) ions are actively gated by voltage-dependent calcium channels (VDCC) in response to glucose metabolism, little is known on its specificity in vivo for quantification of islet ß-cell function using Mn and magnetic resonance imaging (MRI). On the other hand, glucagon-like-peptide-1 receptor (GLP-1R) represents a validated target for the estimation of ß-cell mass using radiolabeled exendin-4 (Ex4) and positron emission tomography (PET). However, a multiparametric imaging workflow revealing ß-cell mass and function quantitatively is still missing. Methods: We developed a simultaneous PET/MRI protocol to comprehensively quantify in vivo changes in ß-cell mass and function by targeting, respectively, GLP-1R and VDCC coupled with insulin secretion. Differences in the spatial distribution of Mn and radiolabeled Ex4 were monitored overtime in native and transgenic pancreata, characterized by spontaneous pancreatic neuroendocrine tumor development. Follow-up with mass spectrometry imaging (MSI) and autoradiography allowed the ex vivo validation of the specificity of Mn and PET tracer uptake and the detection of endogenous biometals, such as calcium and zinc, throughout the endocrine and exocrine pancreas. Results: Our in vivo data based on a volumetric PET/MRI readout for native pancreata and insulinomas connects uptake of Mn measured at early imaging time points to high non-specific binding by the exocrine tissue, while specific retention was only found 24 h post injection. These results are supported by cross-validation of the spatial distribution of exogenous 55Mn and endogenous 44Ca and 64Zn as well with the specific internalization of the radiolabeled peptide targeting GLP-1R. Conclusion: Simultaneous PET/MR imaging of the pancreas enabled the comprehensive in vivo quantification of ß-cell function and mass using Mn and radiolabeled Ex4. Most important, our data revealed that only late time-point measurements reflect the Mn uptake in the islet ß-cells, while early time points detect non-specific accumulation of Mn in the exocrine pancreas.

Regulation of calcineurin activity in insulin-secreting cells: stimulation by Hsp90 during glucocorticoid-induced apoptosis.

Previously, we described that apoptotic cell death induced by the synthetic glucocorticoid dexamethasone (dex) is inhibited by calcineurin inhibitors, FK506 and deltamethrin, in insulin-secreting cells. The aim of the present study was to examine the mechanism of dex-dependent activation of calcineurin. In INS-1 cells cultured up to 4d with dex (100 nmol/l), the percentage of apoptosis, quantified by condensed nuclei and TUNEL positive cells, increased from 1% to 10.9%. FK506 inhibited dex-mediated cell death. Apoptosis was significantly higher at glucose concentrations that induce [Ca(2+)](i) oscillations than at low, non-stimulatory glucose. Dex had no acute effect on [Ca(2+)](i). Calcineurin activity, measured in control and dex-treated cell homogenates, revealed that maximal activity and the sensitivity to the substrate RII peptide was unaltered. However, dex treatment significantly increased enzyme activity at submaximal, physiological Ca(2+) concentrations. Dex did not stimulate the Ca(2+)-dependent protease calpain, known to activate calcineurin by cleavage, as no cleaved calcineurin was detectable. Furthermore, the calpain inhibitor ALLN did not counteract dex-dependent cell death. Western blotting revealed that in dex-treated cells heat shock protein 90 (Hsp90), a component of the glucocorticoid receptor (GR) known to stimulate calcineurin, was increased while calcineurin protein levels were unchanged. In immunoprecipitates with calcineurin antibodies, Hsp90 was only detected in dex-treated cell homogenates. These data suggest that dex-induced apoptosis involves release of Hsp90 from the stimulated GR complex, subsequent binding to and activation of calcineurin, that may contribute to dex-mediated cell death in the presence of high glucose.

ATP mediates a negative autocrine signal on stimulus-secretion coupling in mouse pancreatic ß-cells.

PURPOSE: The role of ATP, which is secreted by pancreatic ß-cells, is still a matter of debate. It has been postulated that extracellular ATP acts as a positive auto- or paracrine signal in ß-cells amplifying insulin secretion. However, there is rising evidence that extracellular ATP may also mediate a negative signal. METHODS: We evaluated whether extracellular ATP interferes with the Ca2+-mediated negative feedback mechanism that regulates oscillatory activity of ß-cells. RESULTS: To experimentally uncover the Ca2+-induced feedback we applied a high extracellular Ca2+ concentration. Under this condition ATP (100 µM) inhibited glucose-evoked oscillations of electrical activity and hyperpolarized the membrane potential. Furthermore, ATP acutely increased the interburst phase of Ca2+ oscillations and reduced the current through L-type Ca2+ channels. Accordingly, ATP (500 µM) decreased glucose-induced insulin secretion. The ATP effect was not mimicked by AMP, ADP, or adenosine. The use of specific agonists and antagonists and mice deficient of large conductance Ca2+-dependent K+ channels revealed that P2X, but not P2Y receptors, and Ca2+-dependent K+ channels are involved in the underlying signaling cascade induced by ATP. The effectiveness of ATP to interfere with parameters of stimulus-secretion coupling is markedly reduced at low extracellular Ca2+ concentration. CONCLUSION: It is suggested that extracellular ATP which is co-secreted with insulin in a pulsatile manner during glucose-stimulated exocytosis provides a negative feedback signal driving ß-cell oscillations in co-operation with Ca2+ and other signals.

TGR5 Activation Promotes Stimulus-Secretion Coupling of Pancreatic ß-Cells via a PKA-Dependent Pathway.

The Takeda-G-protein-receptor-5 (TGR5) mediates physiological actions of bile acids. Since it was shown that TGR5 is expressed in pancreatic tissue, a direct TGR5 activation in ß-cells is currently postulated and discussed. The current study reveals that oleanolic acid (OLA) affects murine ß-cell function by TGR5 activation. Both a Gαs inhibitor and an inhibitor of adenylyl cyclase (AC) prevented stimulating effects of OLA. Accordingly, OLA augmented the intracellular cAMP concentration. OLA and two well-established TGR5 agonists, RG239 and tauroursodeoxycholic acid (TUDCA), acutely promoted stimulus-secretion coupling (SSC). OLA reduced KATP current and elevated current through Ca2+ channels. Accordingly, in mouse and human ß-cells, TGR5 ligands increased the cytosolic Ca2+ concentration by stimulating Ca2+ influx. Higher OLA concentrations evoked a dual reaction, probably due to activation of a counterregulating pathway. Protein kinase A (PKA) was identified as a downstream target of TGR5 activation. In contrast, inhibition of phospholipase C and phosphoinositide 3-kinase did not prevent stimulating effects of OLA. Involvement of exchange protein directly activated by cAMP 2 (Epac2) or farnesoid X receptor (FXR2) was ruled out by experiments with knockout mice. The proposed pathway was not influenced by local glucagon-like peptide 1 (GLP-1) secretion from α-cells, shown by experiments with MIN6 cells, and a GLP-1 receptor antagonist. In summary, these data clearly demonstrate that activation of TGR5 in ß-cells stimulates insulin secretion via an AC/cAMP/PKA-dependent pathway, which is supposed to interfere with SSC by affecting KATP and Ca2+ currents and thus membrane potential.

Dexamethasone induces cell death in insulin-secreting cells, an effect reversed by exendin-4.

Glucocorticoid excess induces hyperglycemia, which may result in diabetes. The present experiments explored whether glucocorticoids trigger apoptosis in insulin-secreting cells. Treatment of mouse beta-cells or INS-1 cells with the glucocorticoid dexamethasone (0.1 micromol/l) over 4 days in cell culture increased the number of fractionated nuclei from 2 to 7 and 14%, respectively, an effect that was reversed by the glucocorticoid receptor antagonist RU486 (1 micromol/l). In INS-1 cells, dexamethasone increased the number of transferase-mediated dUTP nick-end labeling-staining positive cells, caspase-3 activity, and poly-(ADP-) ribose polymerase protein cleavage; decreased Bcl-2 transcript and protein abundance; dephosphorylated the proapoptotic protein of the Bcl-2 family (BAD) at serine155; and depolarized mitochondria. Dexamethasone increased PP-2B (calcineurin) activity, an effect abrogated by FK506. FK506 (0.1 micromol/l) and another calcineurin inhibitor, deltamethrin (1 micromol/l), attenuated dexamethasone-induced cell death. The stable glucagon-like peptide 1 analog, exendin-4 (10 nmol/l), inhibited dexamethasone-induced apoptosis in mouse beta-cells and INS-1 cells. The protective effect of exendin-4 was mimicked by forskolin (10 micromol/l) but not mimicked by guanine nucleotide exchange factor with the specific agonist 8CPT-Me-cAMP (50 micromol/l). Exendin-4 did not protect against cell death in the presence of cAMP-dependent protein kinase (PKA) inhibition by H89 (10 micromol/l) or KT5720 (5 micromol/l). In conclusion, glucocorticoid-induced apoptosis in insulin-secreting cells is accompanied by a downregulation of Bcl-2, activation of calcineurin with subsequent dephosphorylation of BAD, and mitochondrial depolarization. Exendin-4 protects against glucocorticoid-induced apoptosis, an effect mimicked by forskolin and reversed by PKA inhibitors.

Atrial Natriuretic Peptide Affects Stimulus-Secretion Coupling of Pancreatic ß-Cells.

Atrial natriuretic peptide (ANP) influences glucose homeostasis and possibly acts as a link between the cardiovascular system and metabolism, especially in metabolic disorders like diabetes. The current study evaluated effects of ANP on ß-cell function by the use of a ß-cell-specific knockout of the ANP receptor with guanylate cyclase activity (ßGC-A-KO). ANP augmented insulin secretion at the threshold glucose concentration of 6 mmol/L and decreased KATP single-channel activity in ß-cells of control mice but not of ßGC-A-KO mice. In wild-type ß-cells but not ß-cells lacking functional KATP channels (SUR1-KO), ANP increased electrical activity, suggesting no involvement of other ion channels. At 6 mmol/L glucose, ANP readily elicited Ca2+ influx in control ß-cells. This effect was blunted in ß-cells of ßGC-A-KO mice, and the maximal cytosolic Ca2+ concentration was lower. Experiments with inhibitors of protein kinase G (PKG), protein kinase A (PKA), phosphodiesterase 3B (PDE3B), and a membrane-permeable cyclic guanosine monophosphate (cGMP) analog on KATP channel activity and insulin secretion point to participation of the cGMP/PKG and cAMP/PKA/Epac (exchange protein directly activated by cAMP) directly activated by cAMP Epac pathways in the effects of ANP on ß-cell function; the latter seems to prevail. Moreover, ANP potentiated the effect of glucagon-like peptide 1 (GLP-1) on glucose-induced insulin secretion, which could be caused by a cGMP-mediated inhibition of PDE3B, which in turn reduces cAMP degradation.

The LXR Ligand T0901317 Acutely Inhibits Insulin Secretion by Affecting Mitochondrial Metabolism.

The role of liver X receptor (LXR) in pancreatic ß-cell physiology and pathophysiology is still unclear. It has been postulated that chronic LXR activation in ß-cells induces lipotoxicity, a key step in the development of ß-cell dysfunction, which accompanies type 2 diabetes mellitus. In most of these studies, the LXR ligand T0901317 has been administered chronically in the micromolar range to study the significance of LXR activation. In the current study, we have evaluated acute effects of T0901317 on stimulus-secretion coupling of ß-cells. We found that 10 µM T0901317 completely suppressed oscillations of the cytosolic Ca2+ concentration induced by 15 mM glucose. Obviously, this effect was due to inhibition of mitochondrial metabolism. T0901317 markedly depolarized the mitochondrial membrane potential, thus inhibiting adenosine triphosphate (ATP) production and reducing the cytosolic ATP concentration. This led in turn to a huge increase in KATP current and hyperpolarization of the cell membrane potential. Eventually, T0901317 inhibited glucose-induced insulin secretion. These effects were rapid in on-set and not compatible with the activation of a nuclear receptor. In vivo, T0901317 acutely increased the blood glucose concentration after intraperitoneal application. In summary, these data clearly demonstrate that T0901317 exerts acute effects on stimulus-secretion coupling. This observation questions the chronic use of T0901317 and limits the interpretation of results obtained under these experimental conditions.