Rat Beta-cells Research Articles

Role of Na/Ca Exchange and the Plasma Membrane Ca2+–ATPase in β Cell Function and Death

Recent progresses concerning the Na/Ca exchanger (NCX) and the plasma membrane Ca2+-ATPase (PMCA) in the pancreatic beta cell are reviewed. The rat beta cell expresses two splice variants of NCX1 and six splice variants of the 4 PMCA isoforms. At the protein level, the most abundant forms are PMCA2 and PMCA3, providing the first evidence for the presence of these two isoforms in a non-neuronal tissue. Overexpression of NCX1 in an insulinoma cell line altered the initial rise in cytosolic-free Ca2+ concentration ([Ca2+]i) induced by membrane depolarization and the return of the [Ca2+]i to the baseline value on membrane repolarization, indicating that NCX contributes to both Ca2+ inflow and outflow in the beta cell. In contrast, overexpression of the PMCA markedly reduced the global rise in Ca2+ induced by membrane depolarization, indicating that the PMCA has a capacity higher than expected to extrude Ca2+. Glucose, the main physiological stimulus of insulin release from the beta cell, has opposite effect on NCX and PMCA transcription, expression and activity, inducing an increase in the case of NCX and a decrease in the case of the PMCA. This indicates that when exposed to glucose, the beta cell switches from a low-efficiency Ca2+ extruding mechanism, the PMCA, to a high-capacity system, the NCX, in order to better face the increase in Ca2+ inflow induced by the sugar. To our knowledge, this is the first demonstration of a reciprocal change in PMCA and NCX1 expression and activity in response to a given stimulus in any tissue.

Transcriptional profiling of type 1 diabetes genes on chromosome 21 in a rat beta-cell line and human pancreatic islets

We recently finemapped a type 1 diabetes (T1D)-linked region on chromosome 21, indicating that one or more T1D-linked genes exist in this region with 33 annotated genes. In the current study, we have taken a novel approach using transcriptional profiling in predicting and prioritizing the most likely candidate genes influencing beta-cell function in this region. Two array-based approaches were used, a rat insulinoma cell line (INS-1alphabeta) overexpressing pancreatic duodenum homeobox 1 (pdx-1) and treated with interleukin 1beta (IL-1beta) as well as human pancreatic islets stimulated with a mixture of cytokines. Several candidate genes with likely functional significance in T1D were identified. Genes showing differential expression in the two approaches were highly similar, supporting the role of these specific gene products in cytokine-induced beta-cell damage. These were genes involved in cytokine signaling, oxidative phosphorylation, defense responses and apoptosis. The analyses, furthermore, revealed several transcription factor binding sites shared by the differentially expressed genes and by genes demonstrating highly similar expression profiles with these genes. Comparable findings in the rat beta-cell line and human islets support the validity of the methods used and support this as a valuable approach for gene mapping and identification of genes with potential functional significance in T1D, within a region of linkage.

In silico identification of NF-kappaB-regulated genes in pancreatic beta-cells

BackgroundPancreatic beta-cells are the target of an autoimmune attack in type 1 diabetes mellitus (T1DM). This is mediated in part by cytokines, such as interleukin (IL)-1β and interferon (IFN)-γ. These cytokines modify the expression of hundreds of genes, leading to beta-cell dysfunction and death by apoptosis. Several of these cytokine-induced genes are potentially regulated by the IL-1β-activated transcription factor (TF) nuclear factor (NF)-κB, and previous studies by our group have shown that cytokine-induced NF-κB activation is pro-apoptotic in beta-cells. To identify NF-κB-regulated gene networks in beta-cells we presently used a discriminant analysis-based approach to predict NF-κB responding genes on the basis of putative regulatory elements.ResultsThe performance of linear and quadratic discriminant analysis (LDA, QDA) in identifying NF-κB-responding genes was examined on a dataset of 240 positive and negative examples of NF-κB regulation, using stratified cross-validation with an internal leave-one-out cross-validation (LOOCV) loop for automated feature selection and noise reduction. LDA performed slightly better than QDA, achieving 61% sensitivity, 91% specificity and 87% positive predictive value, and allowing the identification of 231, 251 and 580 NF-κB putative target genes in insulin-producing INS-1E cells, primary rat beta-cells and human pancreatic islets, respectively. Predicted NF-κB targets had a significant enrichment in genes regulated by cytokines (IL-1β or IL-1β + IFN-γ) and double stranded RNA (dsRNA), as compared to genes not regulated by these NF-κB-dependent stimuli. We increased the confidence of the predictions by selecting only evolutionary stable genes, i.e. genes with homologs predicted as NF-κB targets in rat, mouse, human and chimpanzee.ConclusionThe present in silico analysis allowed us to identify novel regulatory targets of NF-κB using a supervised classification method based on putative binding motifs. This provides new insights into the gene networks regulating cytokine-induced beta-cell dysfunction and death.

Insulin-induced immunohistochemical and morphological changes in pancreatic beta-cells of streptozotocin-treated diabetic rats

This study was undertaken to investigate insulin-induced changes in the immunohistochemistry and morphometry of pancreatic beta-cells, plasma insulin and blood glucose concentrations of streptozotocin (STZ)-treated diabetic rats. Fifty male Wistar rats (200-250 g) were randomly divided into three experimental groups (viz., A: control group, B: STZ-treated group, and C: STZ+insulin-treated group). Diabetes was induced in group B and group C rats by single intraperitoneal injections of STZ (75 mg/kg body weight), while each animal in the "control" group A received equal volume of citrate buffer solution (pH 6.3) intraperitoneally. STZ+insulin-treated group C diabetic rats were additionally treated with subcutaneous injections of lente insulin (0.5 U/kg body weight) daily from Day 10 to Day 30 of our 40-day study period. The rats used were sacrificed at different time intervals (10th, 20th, 30th and up to the 40th day) following STZ treatment. Fragments of endocrine pancreas of each rat were randomly processed for immunohistochemistry staining and pancreatic insulin content. In diabetic state, pancreatic beta-cells showed a weak immunostaining for insulin on Day 10. Thereafter, insulin administration (in the group C rats) caused a significant decrease (p < 0.05) in the elevated blood glucose levels, and a significant increase (p < 0.05) in the serum insulin concentrations. The surviving beta-cells regenerated and virtually regained their normal immunostaining and functional status for insulin. On the 30th day, the pancreatic insulin contents of the insulin-treated group C rats showed approximately 45-fold increase in immunoreactivity when compared with the immunoreactivity of the same STZ+insulin-treated rats on Day 10 of the 40-day study period. The present study illustrates the sequence of morphological changes that occur in the islets of Langerhans following STZ administration and subsequent insulin treatment. The study also suggests that administration of a moderate single dose of STZ in Wistar rats produces specific necrosis of beta-cells, typical of type 1 insulin-dependent diabetes. The experimental evidence obtained in this study appears to suggest that induction of regenerative stimulus (by insulin treatment) in diabetic state triggers pancreatic regenerative processes, thereby restoring functional activities of the pancreas.

The neurosecretory vesicle protein phogrin is a phosphatidylinositol phosphatase whose activity regulates insulin secretion

Phogrin (NE-6, IA-2β) is a 64KD protein present on insulin-containing secretory granules in pancreatic beta cells. Auto-antibodies against Phogrin are common in pre-diabetics and are used clinically to diagnose a pre-diabetic state. Although Phogrin has sequence homology to tyrosine phosphatases, no enzymatic activity has been reported. We therefore tested Phogrin for enzymatic activity, determined its preferred substrates and tested for a possible role in insulin secretion. We found that Phogrin dephosphorylates inositol phospholipids, including phosphatidylinositol–4,5-bisphospate, PI(4,5)P2, which is known to regulate membrane vesicle dynamics during insulin secretion. Additionally, PKA-dependent phosphorylation of Phogrin decreases its phosphatidylinositol phosphatase activity by 80%. Phogrin overexpression in rat beta cells reduced plasma membrane levels of PI(4,5)P2 by 50% and decreased glucose stimulated insulin secretion by 80%. Our results suggest that Phogrin is a phosphatidylinositol phosphatase that contributes to differential expression of PI(4,5)P2 in secretory vesicles and the plasma membrane and thereby helps regulate insulin secretion. These results identify steps at which the secretory pathway for insulin release may be mis-regulated in diabetes and/or manipulated therapeutically. Source of funding: JDRF 1-2006-841, NIH T32 HL07312.

Expression Analysis of cPLA2 Alpha Interacting TIP60 in Diabetic KKAy and Non-Diabetic C57BL Wild-Type Mice: No Impact of Transient and Stable TIP60 Overexpression on Glucose-Stimulated Insulin Secretion in Pancreatic Beta-Cells

In the present study we investigate the expression levels of cytosolic phospholipase A2 alpha (cPLA2alpha) interacting histone acetyl transferase proteins TIP60alpha and TIP60beta in non-diabetic C57BL wild-type mice and obese type 2 diabetic KKAy model mice. The aim was to test our hypothesis that TIP60 plays a regulatory role in glucose-stimulated insulin secretion from pancreatic beta-cells. Ten obese diabetic KKAy mice and ten non-diabetic C57BL mice were fed a standard chow diet. After nine weeks, islet RNA was purified and used to measure TIP60 expression. We investigated the effect of TIP60alpha and TIP60beta on glucose-stimulated insulin secretion by transient and stable overexpression in the pancreatic mouse beta-cell line MIN6 and the rat beta-cell line INS-1E. We found that non-diabetic C57BL mice and diabetic KKAy mice have the same level of both the alpha and beta splice forms of TIP60. Furthermore, we demonstrated that transient and stable expression of TIP60 in INS-1E cells affects neither glucose-stimulated insulin secretion, insulin output nor cell insulin content. Also susceptibility to developing gluco-toxicity was unaffected. TIP60 over-expression does not affect glucose stimulated insulin secretion, insulin content or abnormal beta-cell function during glucotoxicity.

Tissue-specific transcription factor HNF4α inhibits cell proliferation and induces apoptosis in the pancreatic INS-1 β-cell line

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue-specific transcription factor expressed in many cell types, including pancreatic beta-cells. Mutations in the HNF4alpha gene in humans give rise to maturity-onset diabetes of the young (MODY1) characterized by defective insulin secretion by beta-cells. To elucidate the mechanism underlying this disease, we introduced the splice form HNF4alpha2 or HNF4alpha8 into the rat beta-cell line INS-1. Upon tetracycline-induced expression, both HNF4alpha isoforms caused distinct changes in cell morphology and a massive loss of cell numbers that was correlated with reduced proliferation and induced apoptosis. This differential activity was reflected in oligonucleotide microarray analysis that identified more genes affected by HNF4alpha2 compared to HNF4alpha8, and suggests that both isoforms regulate largely the same set of genes, with HNF4alpha2 being a stronger transactivator. We verified the induction of selected transcripts by real-time RT-PCR, including KAI1 and AIF, both known to have apoptotic potential. By establishing cell lines with inducible expression of these target genes, we deduce that both factors are insufficient to induce apoptosis. We propose that the anti-proliferative and apoptotic properties of HNF4alpha may be an essential feature impaired in MODY1 and possibly also in type 2 diabetes.

Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Pancreatic β-Cells

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels mediate the pacemaker current (Ih or If) observed in electrically rhythmic cardiac and neuronal cells. Here we describe a hyperpolarization-activated time-dependent cationic current, beta-Ih, in pancreatic beta-cells. Transcripts for HCN1-4 were detected by RT-PCR and quantitative PCR in rat islets and MIN6 mouse insulinoma cells. beta-Ih in rat beta-cells and MIN6 cells displayed biophysical and pharmacological properties similar to those of HCN currents in cardiac and neuronal cells. Stimulation of cAMP production with forskolin/3-isobutyl-1-methylxanthine (50 microM) or dibutyryl-cAMP (1 mM) caused a significant rightward shift in the midpoint activation potential of beta-Ih, whereas expression of either specific small interfering (si)RNA against HCN2 (siHCN2b) or a dominant-negative HCN channel (HCN1-AAA) caused a near-complete inhibition of time-dependent beta-Ih. However, expression of siHCN2b in MIN6 cells had no affect on glucose-stimulated insulin secretion under normal or cAMP-stimulated conditions. Blocking beta-Ih in intact rat islets also did not affect membrane potential behavior at basal glucose concentrations. Taken together, our experiments provide the first evidence for functional expression of HCN channels in the pancreatic beta-cell.

GLP-1 receptor activation improves β cell function and survival following induction of endoplasmic reticulum stress

Perturbation of endoplasmic reticulum (ER) homeostasis impairs insulin biosynthesis, beta cell survival, and glucose homeostasis. We show that a murine model of diabetes is associated with the development of ER stress in beta cells and that treatment with the GLP-1R agonist exendin-4 significantly reduced biochemical markers of islet ER stress in vivo. Exendin-4 attenuated translational downregulation of insulin and improved cell survival in purified rat beta cells and in INS-1 cells following induction of ER stress in vitro. GLP-1R agonists significantly potentiated the induction of ATF-4 by ER stress and accelerated recovery from ER stress-mediated translational repression in INS-1 beta cells in a PKA-dependent manner. The effects of exendin-4 on the induction of ATF-4 were mediated via enhancement of ER stress-stimulated ATF-4 translation. Moreover, exendin-4 reduced ER stress-associated beta cell death in a PKA-dependent manner. These findings demonstrate that GLP-1R signaling directly modulates the ER stress response leading to promotion of beta cell adaptation and survival.

Release of small transmitters through kiss-and-run fusion pores in rat pancreatic β cells

Exocytosis of secretory vesicles begins with a fusion pore connecting the vesicle lumen to the extracellular space. This pore may then expand or it may close to recapture the vesicle intact. The contribution of the latter, termed kiss-and-run, to exocytosis of pancreatic beta cell large dense-core vesicles (LDCVs) is controversial. Examination of single vesicle fusion pores demonstrated that rat beta cell LDCVs can undergo exocytosis by rapid pore expansion, by the formation of stable pores, or via small transient kiss-and-run fusion pores. Elevation of cAMP shifted LDCV fusion pore openings to the transient mode. Under this condition, the small fusion pores were sufficient for release of ATP, stored within LDCVs together with insulin. Individual ATP release events occurred coincident with amperometric "stand alone feet" representing kiss-and-run. Therefore, the LDCV kiss-and-run fusion pores allow small transmitter release but likely retain the larger insulin peptide. This may represent a mechanism for selective intraislet signaling.

Effect of cerebrocrast, a new long‐acting compound on blood glucose and insulin levels in rats when administered before and after STZ‐induced diabetes mellitus

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that is characterized by selective destruction of insulin secreting pancreatic islets beta-cells. The formation of cytokines (IL-1beta, IL-6, TNF-alpha, etc.) leads to extensive morphological damage of beta-cells, DNA fragmentation, decrease of glucose oxidation, impaired glucose-insulin secretion and decreased insulin action and proinsulin biosynthesis. We examined the protective effect of a 1,4-dihydropyridine (DHP) derivative cerebrocrast (synthesized in the Latvian Institute of Organic Synthesis) on pancreatic beta-cells in rats possessing diabetes induced with the autoimmunogenic compound streptozotocin (STZ). Cerebrocrast administration at doses of 0.05 and 0.5 mg/kg body weight (p.o.) 1 h or 3 days prior to STZ as well as at 24 and 48 h after STZ administration partially prevented pancreatic beta-cells from the toxic effects of STZ, and delayed the development of hyperglycaemia. Administration of cerebrocrast starting 48 h after STZ-induced diabetes in rats for 3 consecutive days at doses of 0.05 and 0.5 mg/kg body weight (p.o.) significantly decreased blood glucose level, and the effect remained 10 days after the last administration. Moreover, in these rats, cerebrocrast evoked an increase of serum immunoreactive insulin (IRI) level during 7 diabetic days as compared to both the control normal rats and the STZ-induced diabetic control rats. The STZ-induced diabetic rats that received cerebrocrast had a significantly high serum IRI level from the 14th to 21st diabetic days in comparison with the STZ-induced diabetic control. The IRI level in serum as well as the glucose disposal rate were significantly increased after stimulation of pancreatic beta-cells with glucose in normal rats that received cerebrocrast, administered 60 min before glucose. Glucose disposal rate in STZ-induced diabetic rats as a result of cerebrocrast administration was also increased in comparison with STZ-diabetic control rats. Administration of cerebrocrast in combination with insulin intensified the effect of insulin. The hypoglycaemic effect of cerebrocrast primarily can be explained by its immunomodulative properties. Moreover, cerebrocrast can act through extrapancreatic mechanisms that favour the expression of glucose transporters, de novo insulin receptors formation in several cell membranes as well as glucose uptake.

Involvement of Ca 2+, CaMK II and PKA in EGb 761-induced insulin secretion in INS-1 cells

EGb 761, a standardized form of Ginkgo biloba L. (Ginkgoaceae) leaf extract, was recently reported to increase pancreatic beta-cell function. To determine whether EGb 761 elicits insulin secretion directly, we treated INS-1 rat beta cells with EGb 761 and then measured insulin release. Treatment of EGb 761 (50 μg/ml) significantly stimulated insulin secretion in INS-1 cells, compared with untreated control ( p < 0.05) and the stimulatory effect of EGb 761 on insulin secretion was dose-dependent. To elucidate the mechanism of EGb 761-induced insulin secretion, we investigated the involvement of calcium. The treatment with nifedipine, an L-type calcium channel blocker, prevented EGb 761-induced insulin secretion and furthermore, EGb 761 itself elevated [Ca 2+] i, suggesting the involvement of calcium in this process. To identity the protein kinases involved in EGb 761-induced insulin secretion, INS-1 cells were treated with different kinase inhibitors and their effects on EGb 761-induced secretion were investigated. KN62 and H89, calium/calmodulin kinase (CaMK) II and protein kinase A (PKA) inhibitor, respectively, significantly reduced EGb 761-induced insulin secretion. Immunoblotting studies showed an increase in the phosphorylated-forms of CaMK II and of PKA substrates after EGb 761 treatment. Our data suggest that EGb 761-induced insulin secretion is mediated by [Ca 2+] i elevation and subsequent activation of CaMK II and PKA.

IL-1β-induced pro-apoptotic signalling is facilitated by NCAM/FGF receptor signalling and inhibited by the C3d ligand in the INS-1E rat beta cell line

IL-1beta released from immune cells induces beta cell pro-apoptotic signalling via mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB). In neurons, the neural cell adhesion molecule (NCAM) signals to several elements involved in IL-1beta-induced pro-apoptotic signalling in beta cells. Pancreatic beta cells express NCAM, but its biological effects in these cells are unclear. The aim of this study was to investigate whether there is cross-talk between NCAM signalling and cytokine-induced pro-apoptotic signalling. Western blotting was used to investigate levels of NCAM and inducible nitric oxide synthase, phosphorylation of Src and MAPKs, and cleavage of caspase-3. MAPK activity was investigated with an in vitro kinase assay. Apoptosis was detected by cleaved caspase-3 and a Cell Death Detection ELISA(plus) assay. NCAM-induced fibroblast growth factor receptor (FGFR) activation was investigated in NCAM(-/-) Trex293 cells where FGFR phosphorylation was measured by Western blotting after NCAM transfection. Pre-exposure of INS-1E cells to the FGFR-inhibitor SU5402, but not to the Src-inhibitor PP2, dose-dependently inhibited IL-1beta-mediated MAPK activity. A synthetic peptide, C3d, reported to bind NCAM, did not activate MAPK or Akt as reported in neurons but inhibited IL-1beta-induced MAPK activity, thereby mimicking the effect of SU5402. Furthermore, C3d inhibited NCAM-induced FGFR phosphorylation and apoptosis induced by IL-1beta plus IFN-gamma, but did not affect IL-1beta-induced NF-kappaB signalling. We suggest that NCAM signalling through FGFR is required for efficient IL-1beta pro-apoptotic signalling by facilitating IL-1beta-induced MAPK activation downstream of the NF-kappaB-MAPK branching point. Further, these data identify a novel function of C3d as an inhibitor of NCAM-induced FGFR activity and of IL-1beta-induced MAPK activation in beta cells.

Ghrelin prevents development of diabetes at adult age in streptozotocin-treated newborn rats

Ghrelin, a stomach-derived hormone, functions in multiple biological processes, including glucose metabolism and cellular differentiation and proliferation. In this study, we examined whether early treatment with ghrelin can regenerate beta cells of the pancreas in an animal model of diabetes mellitus, the n0-STZ model, in which neonatal rats are injected with streptozotocin (STZ) at birth. Following administration of ghrelin to n0-STZ rats from postnatal days 2 to 8, we examined beta cell mass, mRNA expression levels of insulin and of pancreatic and duodenal homeobox 1 (Pdx1) gene, and pancreatic morphology on days 21 and 70. In addition, we investigated the effects of ghrelin on beta cell replication. By day 21, ghrelin treatment increased pancreatic expression of insulin and Pdx1 mRNA in n0-STZ rats. The number of replicating cells was also significantly increased in the ghrelin-treated n0-STZ model. At day 70, n0-STZ rats exhibited hyperglycaemia, despite slight increases in plasma insulin levels. Ghrelin treatment resulted in the improvement of plasma glucose levels, which were associated with normal plasma insulin levels. Pancreatic insulin mRNA and protein levels were significantly increased in ghrelin-treated n0-STZ model animals. These findings suggest that ghrelin promotes regeneration of beta cells in STZ-treated newborn rats. Thus, early administration of ghrelin may help prevent the development of diabetes in disease-prone subjects after beta cell destruction.

Calbindin-D 28k decreases L-type calcium channel activity and modulates intracellular calcium homeostasis in response to K + depolarization in a rat beta cell line RINr1046-38

Calbindin-D 28k, acts as a modulator of depolarization induced calcium transients in the pancreatic beta cell. However, specific mechanisms have not been defined. Here we show for the first time that the calcium binding protein calbindin-D 28k acts by affecting calcium influx through voltage-dependent calcium channels in RIN pancreatic beta cells. Whole-cell patch–clamp recordings revealed that Ca 2+ current amplitudes of calbindin-D 28k expressing RINr1046-38 beta cells were smaller than the Ca 2+ current amplitudes in control cells in response to depolarizing pulses. The peak current was observed at +20 mV and the average amplitude was ∼50 pA in the calbindin expressing cells compared to ∼250 pA in control cells. In calbindin-D 28k expressing cells, the channels had enhanced sensitivity to Ca 2+ dependent inactivation and currents decayed much more rapidly than in control cells. The Ca 2+ channels affected by calbindin were found to have biophysical properties consistent with dihydropyridine-sensitive L-type calcium channels. In response to depolarizing concentrations of K +, calbindin expression caused a five-fold decrease in the rate of rise of [Ca 2+] i and decay was slower in the calbindin expressing cells. Application of verapamil resulted in a drop in the [Ca 2+] i signal to pre-stimulation levels indicating that the Ca 2+ channel responsible for the depolarization evoked Ca 2+ entry, modulated by calbindin, is the L-type. Co-immunoprecipitation and GST pull-down assays indicate that calbindin-D 28k can interact with the α 1 subunit of Ca v1.2. We thus conclude that calbindin-D 28k can regulate calcium influx via L-type calcium channels. Our findings suggest a role for calbindin-D 28k in the beta cell in modulating Ca 2+ influx via L-type voltage-dependent calcium channels.

Fatty acid-induced toxicity and neutral lipid accumulation in insulin-producing RINm5F cells

Fatty acids have been shown to cause death of rat and human primary pancreatic beta cells and of insulin-producing cell lines. These studies focused mainly on saturated and monounsaturated FA such as palmitic, stearic and oleic acids. In this study, we have performed a comparison of the toxicity of a wider range of FA. The toxicity of different FA to insulin-producing RINm5F cells was assessed by flow cytometry measuring loss of plasma membrane integrity and increase in DNA fragmentation. Additionally, the FA induced neutral lipid accumulation and the FA composition were determined. Palmitic, linoleic, γ-linolenic, oleic, stearic, and eicosapentaenoic acid caused DNA fragmentation of insulin-producing RINm5F cells. Loss of membrane integrity was mainly caused by linoleic and γ-linolenic acid. There was no correlation between cytotoxicity and the abundance of the FA in the cells as determined by HPLC analysis. Taken as whole, the toxic effect of the FA on insulin-producing RINm5F cells varied irrespective of the chain length and the degree of unsaturation. In these cells PA and LA exhibited the highest toxicity, whereas AA was not toxic. In addition, the toxicity of most tested FA was inversely related to low NLA, except for AA and EPA. The results of this study contribute to the understanding of the role of FA in the impairment of pancreatic beta cell function that occurs in type 2 diabetes and obesity.

Extracellular-Added ADP-Ribose Increases Intracellular Free Ca2+ Concentration Through Ca2+ Release From Stores, but Not Through TRPM2-Mediated Ca2+ Entry, in Rat β-Cell Line RIN-5F

Intracellular ADP-ribose is an activator of TRPM2, which is a Ca2+-permeable channel and mediates H2O2-induced cell death, in the TRPM2-expressing rat beta-cell line RIN-5F. We examined the effect of extracellular-added ADP-ribose on intracellular Ca2+ concentration in RIN-5F cells. ADP-ribose induced Ca2+ release from the thapsigargin-sensitive Ca2+ store, but not Ca2+ entry across the plasma membrane. A phospholipase C (PLC) inhibitor and a non-specific IP3 receptor inhibitor blocked its Ca2+ release. H2O2-induced Ca2+ entry through TRPM2 was not affected by extracellular ADP-ribose. These findings suggest that extracellular-added ADP-ribose induces Ca2+ release through the PLC-IP3 pathway and does not act as a TRPM2 activator.

Dopamine D2-like Receptors Are Expressed in Pancreatic Beta Cells and Mediate Inhibition of Insulin Secretion

Dopamine signaling is mediated by five cloned receptors, grouped into D1-like (D1 and D5) and D2-like (D2, D3 and D4) families. We identified by reverse transcription-PCR the presence of dopamine receptors from both families in INS-1E insulin-secreting cells as well as in rodent and human isolated islets. D2 receptor expression was confirmed by immunodetection revealing localization on insulin secretory granules of INS-1E and primary rodent and human beta cells. We then tested potential effects mediated by the identified receptors on beta cell function. Dopamine (10 microM) and the D2-like receptor agonist quinpirole (5 microM) inhibited glucose-stimulated insulin secretion tested in several models, i.e. INS-1E beta cells, fluorescence-activated cell-sorted primary rat beta cells, and pancreatic islets of rat, mouse, and human origin. Insulin exocytosis is controlled by metabolism coupled to cytosolic calcium changes. Measurements of glucose-induced mitochondrial hyperpolarization and ATP generation showed that dopamine and D2-like agonists did not inhibit glucose metabolism. On the other hand, dopamine decreased cell membrane depolarization as well as cytosolic calcium increases evoked by glucose stimulation in INS-1E beta cells. These results show for the first time that dopamine receptors are expressed in pancreatic beta cells. Dopamine inhibited glucose-stimulated insulin secretion, an effect that could be ascribed to D2-like receptors. Regarding the molecular mechanisms implicated in dopamine-mediated inhibition of insulin release, our results point to distal steps in metabolism-secretion coupling. Thus, the role played by dopamine in glucose homeostasis might involve dopamine receptors, expressed in pancreatic beta cells, modulating insulin release.

Glucose‐induced electrical activity in rat pancreatic β‐cells: dependence on intracellular chloride concentration

A rise in glucose concentration depolarizes the beta-cell membrane potential leading to electrical activity and insulin release. It is generally believed that closure of KATP channels underlies the depolarizing action of glucose, though work from several laboratories has indicated the existence of an additional anionic mechanism. It has been proposed that glucose activates a volume-regulated anion channel, generating an inward current due to Cl- efflux. This mechanism requires that intracellular [Cl-] is maintained above its electrochemical equilibrium. This hypothesis was tested in rat beta-cells by varying [Cl-] in the patch pipette solution using the Cl--permeable antibiotic amphotericin B to allow Cl- equilibration with the cell interior. Under such conditions, a depolarization and electrical activity could be evoked by 16 mM glucose with pipette solutions containing 80 or 150 mM Cl-. At 40 or 20 mM Cl-, a subthreshold depolarization was usually observed, whilst further reduction to 12 or 6 mM abolished depolarization, in some cases leading to a glucose-induced hyperpolarization. With a pipette solution containing gramicidin, which forms Cl--impermeable pores, glucose induced a depolarization and electrical activity irrespective of [Cl-] in the pipette solution. Under the latter conditions, glucose-induced electrical activity was prevented by bumetanide, an inhibitor of the Na+-K+-2Cl- co-transporter. This inhibition could be overcome by the use of amphotericin B with a high [Cl-] pipette solution. These findings suggest that the maintenance of high intracellular [Cl-] in the beta-cell is an important determinant in glucose-induced depolarization, and support the hypothesis that beta-cell stimulation by glucose involves activation of the volume-regulated anion channel and generation of an inward Cl- current.

Structure−Function Studies of PANDER, an Islet Specific Cytokine Inducing Cell Death of Insulin-Secreting β Cells

PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretory granules, that induces apoptosis of alpha and beta cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of 4 alpha-helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutated PANDERs were constructed and expressed in beta-TC3 cells to identify the essential region of PANDER involved in beta-cell death. Beta-cell function was assessed by assays of cell viability and insulin secretion. Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in beta-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized and detected in beta-TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncation of helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices C and D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A, the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced beta-cell death. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfide bond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the active part of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased beta-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices B and C and the second disulfide bond of PANDER are essential for PANDER-induced beta-cell death.