Rat Islet Β-cells Research Articles

Antibodies against H1N1 influenza virus hemagglutinin cross-react with prohibitin

Influenza virus infection is associated with type 1 diabetes (T1DM), but its pathogenesis remains unclear. Here, our study found that one of the monoclonal antibodies against H1N1 influenza virus hemagglutinin(HA) cross-reacted with human pancreatic tissue and further demonstrated that it binded to rat islet β-cells. We immunoprecipitated islet protein with this cross-reactive antibody and identified the bound antigen as prohibitin by mass spectrometry. We then expressed the prohibitin protein in bacteria and confirmed the antibody binding to prohibitin by Western blot. We also verified the cross-reactivity of the antibody by prohibitin-siRNA transfection in islet beta cells. We conclude that prohibitin is an autoantigen that cross-reacts with influenza virus HA. The correlation between the autoantigen prohibitin and type 1 diabetes remains to be investigated.

Extracellular CADM1 interactions influence insulin secretion by rat and human islet β-cells and promote clustering of syntaxin-1.

Contact between β-cells is necessary for their normal function. Identification of the proteins mediating the effects of β-cell-to-β-cell contact is a necessary step toward gaining a full understanding of the determinants of β-cell function and insulin secretion. The secretory machinery of the β-cells is nearly identical to that of central nervous system (CNS) synapses, and we hypothesize that the transcellular protein interactions that drive maturation of the two secretory machineries upon contact of one cell (or neural process) with another are also highly similar. Two such transcellular interactions, important for both synaptic and β-cell function, have been identified: EphA/ephrin-A and neuroligin/neurexin. Here, we tested the role of another synaptic cleft protein, CADM1, in insulinoma cells and in rat and human islet β-cells. We found that CADM1 is a predominant CADM isoform in β-cells. In INS-1 cells and primary β-cells, CADM1 constrains insulin secretion, and its expression decreases after prolonged glucose stimulation. Using a coculture model, we found that CADM1 also influences insulin secretion in a transcellular manner. We asked whether extracellular CADM1 interactions exert their influence via the same mechanisms by which they influence neurotransmitter exocytosis. Our results suggest that, as in the CNS, CADM1 interactions drive exocytic site assembly and promote actin network formation. These results support the broader hypothesis that the effects of cell-cell contact on β-cell maturation and function are mediated by the same extracellular protein interactions that drive the formation of the presynaptic exocytic machinery. These interactions may be therapeutic targets for reversing β-cell dysfunction in diabetes.

GW9508 inhibits insulin secretion by activating ATP-sensitive potassium channels in rat pancreatic β-cells

GW9508 is an agonist of G protein-coupled receptor 40 (GPR40) that is expressed in pancreatic β-cells and is reported to regulate insulin secretion. However, the effects of GW9508 on pancreatic β-cells in primary culture have not been well investigated. This study measured the acute effects of GW9508 on insulin secretion from rat pancreatic islets in primary culture, and the insulin secretion-related events such as the changes in membrane potential, ATP-sensitive potassium currents (KATP currents), and intracellular Ca(2+) concentrations ([Ca(2+)]i) of rat islet β-cells were also recorded. GW9508 (10-40 μM) did not influence basal insulin levels at 2 mM glucose, but it (above 20 μM) significantly inhibited 5 and 15 mM glucose-stimulated insulin secretion (GSIS). GW9508 did not inhibit insulin secretion stimulated by tolbutamide, the closer of KATP channels. GW9508 activated KATP channels and blocked the membrane depolarization and the increase in [Ca(2+)]i that were stimulated by glucose. GW9508 itself stimulated a transient increase in [Ca(2+)]i, which was fully blocked by depletion of intracellular Ca(2+) stores with thapsigargin or by inhibition of phospholipase C (PLC) activity with U73122. GW9508-induced activation of KATP channels was only partly inhibited by U73122 treatment. In conclusion, although it stimulates a transient release of Ca(2+) from intracellular Ca(2+) stores via activation of PLC, GW9508 inhibits GSIS by activating KATP channels probably in a distal step to GPR40 activation in rat β-cells.

Tauroursodeoxycholate, a Chemical Chaperone, Prevents Palmitate-induced Apoptosis in Pancreatic β-cells by Reducing ER Stress

Free fatty acids (FFA) can have deleterious effects on β-cells and promote type 2 diabetes, a process known as lipotoxicity. Recently, the induction of endoplasmic reticulum (ER) stress is one mechanism proposed to contribute to the detrimental effects of FFA on β-cells. Tauroursodeoxycholic acid (TUDCA) has been reported to show cytoprotective effects by alleviating ER stress induced by some cytotoxic stimuli. The aim of this study was to investigate the effects of TUDCA on FFA (palmitate)-induced apoptosis and ER stress in rat islet β-cells. The rat pancreatic β-cell line INS-1 was cultured with palmitate (0.5 mM), or cultured togther with TUDCA (100 μM), Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis in INS-1 cells. Cell viability was evaluated with MTT reduction conversion assay. The expressions of ER stress marker GRP78, ER stress-associated pro-apoptotic effectors CHOP and ATF4 were detected by Western blotting. TUDCA significantly reduced palmitate-induced cell apoptosis and growth inhibition in INS-1 cells. TUDCA also attenuated palmitate-induced expressions of GRP78, CHOP and ATF4 in INS-1 cells. Our results thus suggested that TUDCA could protect INS-1 cells from palmitate-induced injury, which might be due to the amelioration of ER stress and blocking the ATF4/CHOP signaling pathway.

Effects of physiological quercetin metabolites on interleukin-1β-induced inducible NOS expression

Cytokines released by inflammatory cells around the pancreatic islets are implicated in the pathogenesis of diabetes mellitus. Specifically, interleukin-1β (IL-1β) is known to be involved in islet β-cell damage by activation of nuclear factor-κB (NF-κB)-mediated inducible nitric oxide synthase (iNOS) gene expression. Though most flavonoids are shown to have various beneficial effects, little is known about the anti-inflammatory effects of their metabolites. Therefore, we investigated the effects of quercetin and its metabolites quercetin 3′-sulfate, quercetin 3-glucuronide and isorhamnetin 3-glucuronide on IL-1β-stimulated iNOS gene expression in RINm5F β-cells. The nitrite level, iNOS protein and its mRNA expression levels and iNOS promoter activity were measured. In addition, IκBα protein phosphorylation, nuclear translocation of nuclear factor-κB (NF-κB) and NF-κB DNA binding activity were determined. Adenosine 5′-triphosphate disodium salt-induced insulin release was also measured. Quercetin significantly reduced IL-1β-induced nitrite production, iNOS protein and its mRNA expression levels, and it also inhibited IL-1β-induced IκBα phosphorylation, NF-κB activation and iNOS promoter activity. Additionally, quercetin significantly restored the inhibition of insulin secretion by IL-1β. Meanwhile, quercetin metabolites did not show any effect on IL-1β-induced iNOS gene expression and also on insulin secretion. Therefore, in terms of iNOS expression mechanism, dietary ingestion of quercetin is unlikely to show anti-inflammatory effects in rat islet β-cells exposed to IL-1β.

Syntaxin-1A Interacts with Distinct Domains within Nucleotide-binding Folds of Sulfonylurea Receptor 1 to Inhibit β-Cell ATP-sensitive Potassium Channels

The ATP-sensitive potassium (K(ATP)) channel regulates pancreatic β-cell function by linking metabolic status to electrical activity. Syntaxin-1A (Syn-1A), a SNARE protein mediating exocytotic fusion, binds and inhibits the K(ATP) channel via the nucleotide-binding folds (NBFs) of its sulfonylurea receptor-1 (SUR1) regulatory subunit. In this study, we elucidated the precise regions within the NBFs required for Syn-1A-mediated K(ATP) inhibition, using in vitro binding assays, whole cell patch clamp and FRET assay. Specifically, NBF1 and NBF2 were each divided into three subregions, Walker A (W(A)), signature sequence linker, and Walker B (W(B)), to make GST fusion proteins. In vitro binding assays revealed that Syn-1A associates with W(A) and W(B) regions of both NBFs. Patch clamp recordings on INS-1 and primary rat β-cells showed that Syn-1A-mediated channel inhibition was reversed by co-addition of NBF1-W(B) (not NBF1-W(A)), NBF2-W(A), and NBF2-W(B). The findings were corroborated by FRET studies showing that these truncates disrupted Syn-1A interactions with full-length SUR1. To further identify the binding sites, series single-site mutations were made in the Walker motifs of the NBFs. Only NBF1-W(A) (K719M) or NBF2-W(A) (K1385M) mutant no longer bound to Syn-1A; K1385M failed to disrupt Syn-1A-mediated inhibition of K(ATP) channels. These data suggest that NBF1-W(A) (Lys-719) and NBF2-W(A) (Lys-1385) are critical for Syn-1A-K(ATP) channel interaction. Taken together, Syn-1A intimately and functionally associates with the SUR1-NBF1/2 dimer via direct interactions with W(A) motifs and sites adjacent to W(B) motifs of NBF1 and NBF2 but transduces its inhibitory actions on K(ATP) channel activity via some but not all of these NBF domains.

Nucleobindin-2/nesfatin in the endocrine pancreas: distribution and relationship to glycaemic state

The protein nucleobindin-2 (NUCB2, also known as nesfatin) was recently implicated as a mediator of anorexia and catabolism in the central nervous system, and has been suggested to act as a cleaved and secreted messenger. Given the overlap of signalling molecules between the brain and pancreas, we have explored the presence of NUCB2 in the islets of Langerhans. We also performed an investigation of the dynamic regulation of pancreatic NUCB2 in different metabolic states. NUCB2-like immunoreactivity was detected by immunofluorescence in all human and rat islet beta-cells (as detected by co-localization with insulin), but not in other islet cells or in the exocrine pancreas. Islet NUCB2 content, as measured by enzyme immunoassay, did not change significantly following an overnight fast, but was substantially lower in islets isolated from an animal model of type 2 diabetes, the Goto-Kakizaki (GK) rats (48% of non-diabetic Wistar rat control). Serum levels, however, were not different between Wistar and GK rats. The release of NUCB2 from isolated rat islets was significantly elevated following glucose challenge (123%), but this effect was substantially lower than that observed for insulin (816%). In contrast, serum levels of NUCB2 showed a reversible decrease in an i.p. glucose tolerance test. These data suggest a role for NUCB2 in beta-cell function and a potential involvement in diabetic pathology. However, our findings, together with previous reports, appear more compatible with intracellular actions rather than with endocrine/paracrine communication, and suggest that NUCB2 in serum derives primarily from non-islet sources.

Cab45b, a Munc18b-interacting Partner, Regulates Exocytosis in Pancreatic β-Cells

Cab45b is a cytosolic Ca(2+)-binding protein reported to regulate zymogen secretion in pancreatic acini. We now show that Cab45b is also expressed in pancreatic islet beta-cells and interacts there with the Sec1-Munc18 protein Munc18b. We employed patch clamp cell capacitance measurements to show that antibodies against Cab45b inhibited depolarization-evoked membrane capacitance increments, suggesting an impact on beta-cell granule exocytosis, both the readily releasable granule pool and refilling of this pool. Site-specific mutants in the Cab45b EF-hands were used to dissect the molecular interactions involved in Cab45b function. Mutants in EF-hands 2 and 3 had no detectable effects on interaction of Cab45b with Munc18b and did not affect the depolarization-evoked calcium currents, but remarkably, they facilitated the complex formation of Munc18b with syntaxin-2 and -3. As a result, these two EF-hand mutants inhibited beta-cell membrane capacitance increments. This inhibition is mediated via Munc18b because Munc18b silencing with small interfering RNA abolished the effects of these two mutants. The results suggest a mechanism for Cab45b action that involves regulating the dynamic association of Munc18b with SNAREs to impact beta-cell granule exocytosis.

Acute activation of acid ceramidase affects cytokine-induced cytotoxicity in rat islet β-cells

Ceramidase hydrolyzes ceramide and produces sphingosine as a substrate of sphingosine kinase (SPHK), which transforms sphingosine to sphingosine-1-phosphate. It has been reported that cytokines elicit SPHK activation in rat β-cells. As a sphingosine provider, ceramidase should also be activated. In our previous work, we showed that the increase in mRNA and protein levels in cytokine-treated INS-1 rat β-cells resulted in chronic activation of neutral ceramidase. Here we found that acid ceramidase (AC) is activated by cytokines at an early stage via tyrosine phosphorylation. In addition, basal AC activity was first detected in INS-1 cells and isolated rat islets, and cytokine-induced cell growth was significantly repressed when AC was pharmacologically inhibited.

SNAP-25 1–180 enhances insulin secretion by blocking Kv2.1 channels in rat pancreatic islet β-cells

Voltage-gated outward K + currents from pancreatic islet β-cells are known to repolarize the action potential during a glucose stimulus, and consequently to modulate Ca 2+ entry and insulin secretion. The voltage gated K + (Kv) channel, Kv2.1, which is expressed in rat islet β-cells, mediates over 60% of the Kv outward K + currents. A novel peptidyl inhibitor of Kv2.1/Kv2.2 channels, guangxitoxin (GxTX)-1, has been shown to enhance glucose-stimulated insulin secretion. Here, we show that SNAP-25 1–180 (S180), an N-terminal SNAP-25 domain, but not SNAP-25 1–206 (S206), inhibits Kv current and enhances glucose-dependent insulin secretion from rat pancreatic islet β-cells, and furthermore, this enhancement was induced by the blockade of the Kv2.1 current. This study indicates that the Kv2.1 channel is a potential target for novel therapeutic agent design for the treatment of type 2 diabetes. This target may possess advantages over currently-used therapies, which modulate insulin secretion in a glucose-independent manner.

Flow Cytometric Quantification of Glucose-Stimulated β-Cell Metabolic Flux Can Reveal Impaired Islet Functional Potency

The objective of this study was to develop a multiparametric flow cytometry assay to simultaneously quantify isolated pancreatic islet cell viability, apoptosis, and glucose-induced metabolic flux. INS-1 and rat islet beta-cells were stained with fluorescent probes for cell viability (ToPro3), apoptosis (Annexin V and VADFMK), and intracellular calcium (Ca2+(i)) (Fura Red), stimulated with glucose, and analyzed on a FACS Vantage flow cytometer. Glucose-induced metabolic activity was indicated by changes in Fura Red fluorescence and the autofluorescence of the pyridine [NAD(P)H] and flavin (FAD/FMN) nucleotides. Rat islets cultured under conditions of proinflammatory cytokine-induced oxidative stress were evaluated by flow cytometry and transplantation into diabetic mice. INS-1 and rat islet beta-cell health and metabolic activity were quantified in response to elevated glucose dose and inhibitors of glycolysis and mitochondrial function. Changes in metabolite fluorescence were converted to an area under the curve (AUC) value. Rat islets cultured under oxidative stress conditions showed decreased viability, increased apoptosis, and decreased glucose-induced metabolic activity indicated by reduced AUC for pyridine and flavin nucleotides and Ca2+(i). Reduced metabolite AUC measured by flow cytometry correlated with the inability to reverse diabetes in mice. Single cell flow cytometry can simultaneously quantify both overall islet cell health and beta-cell glucose responsiveness as indicators of functional potency.

Regulation of the susceptibility to oxidative stress by cysteine availability in pancreatic β-cells

Pancreatic beta-cells are susceptible to oxidative stress, which is related closely to the islet dysfunction. In the present study, using the pancreatic cell lines HIT-T15 and RINm5F as known in vitro models of impaired beta-cell function as well as primary rat islet beta-cells, we observed a relationship between intracellular glutathione levels and oxidative stress-mediated cell dysfunction. Hydrogen peroxide and 4-hydroxy-2-nonenal caused cell death in HIT-T15 and RINm5F cells at lower concentrations compared with non-beta-cells, such as HepG2 and NRK-49F cells. The extent of the cytotoxicity caused by the model oxidants was inversely correlated well with intracellular glutathione levels in the cell lines used. Treatment of HIT-T15 and RINm5F cells with l-cysteine or l-cystine significantly augmented the glutathione contents, surpassing the effect of N-acetylcysteine, and abrogated 4-hydroxy-2-nonenal-mediated cytotoxicity almost completely. l-Cysteine increased intracellular glutathione levels in primary beta-cells as well. Supplementation of l-cysteine to the RINm5F cell culture inhibited 4-hydroxy-2-nonenal-mediated cytosolic translocation of PDX-1, a key transcription factor for beta-cell function. Intrinsic transport activities (V(max)/K(m)) of the l-cystine/l-glutamate exchanger in HIT-T15 and RINm5F cells were considerably lower than that in NRK-49F cells, although gene expressions of the exchanger were similar in these cells. Results obtained from the present study suggest that the restricted activity of the l-cystine/l-glutamate exchanger controls the levels of intracellular glutathione, thereby making beta-cells become susceptible to oxidative stress.

The Actions of a Novel Potent Islet β-Cell–Specific ATP-Sensitive K+ Channel Opener Can Be Modulated by Syntaxin-1A Acting on Sulfonylurea Receptor 1

Islet beta-cell-specific ATP-sensitive K(+) (K(ATP)) channel openers thiadiazine dioxides induce islet rest to improve insulin secretion, but their molecular basis of action remains unclear. We reported that syntaxin-1A binds nucleotide binding folds of sulfonylurea receptor 1 (SUR1) in beta-cells to inhibit K(ATP) channels. As a strategy to elucidate the molecular mechanism of action of these K(ATP) channel openers, we explored the possibility that 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC55-0462) might influence syntaxin-1A-SUR1 interactions or vice versa. Whole-cell and inside-out patch-clamp electrophysiology was used to examine the effects of glutathione S-transferase (GST)-syntaxin-1A dialysis or green fluorescence protein/syntaxin-1A cotransfection on NNC55-0462 actions. In vitro pull-down binding studies were used to examine NNC55-0462 influence on syntaxin-1A-SUR1 interactions. Dialysis of GST-syntaxin-1A into the cell cytoplasm reduced both potency and efficacy of extracellularly perfused NNC55-0462 in a HEK cell line stably expressing Kir6.2/SUR1 (BA8 cells) and in rat islet beta-cells. Moreover, inside-out membrane patches excised from BA8 cells showed that both GST-syntaxin-1A and its H3 domain inhibited K(ATP) channels previously activated by NNC55-0462. This action on K(ATP) channels is isoform-specific to syntaxin-1A because syntaxin-2 was without effect. Furthermore, the parent compound diazoxide showed similar sensitivity to GST-syntaxin-1A inhibition. NNC55-0462, however, did not influence syntaxin-1A-SUR1 binding interaction. Our results demonstrated that syntaxin-1A interactions with SUR1 at its cytoplasmic domains can modulate the actions of the K(ATP) channel openers NNC55-0462 and diazoxide on K(ATP) channels. The reduced levels of islet syntaxin-1A in diabetes would thus be expected to exert a positive influence on the therapeutic effects of this class of K(ATP) channel openers.

Possible role of carbonic anhydrase in rat pancreatic islets: enzymatic, secretory, metabolic, ionic, and electrical aspects

The presence of carbonic anhydrase (type V) was recently documented in rat and mouse pancreatic islet beta-cells by immunostaining and Western blotting. In the present study, the activity of carbonic anhydrase was measured in rat islet homogenates and shown to be about four times lower than in rat parotid cells. The pattern for the inhibitory action of acetazolamide on carbonic anhydrase activity also differed in islet and parotid cell homogenates, suggesting the presence of different isoenzymes. NaN3 inhibited carbonic anhydrase activity in islet homogenates and both D-[U-14C]glucose oxidation and glucose-stimulated insulin secretion. Acetazolamide (0.3-10.0 mM) also decreased glucose-induced insulin output but failed to affect adversely D-[U-14C]glucose oxidation, although it inhibited the conversion of D-[5-3H]glucose to [3H]OH and that of D-[U-14C]glucose to acidic metabolites. Hydrochlorothiazide (3.0-10.0 mM), which also caused a concentration-related inhibition of the secretory response, like acetazolamide (5.0-10.0 mM), decreased H(14)CO3- production from D-[U-14C]glucose (16.7 mM). Acetazolamide (5.0 mM) did not affect the activity of volume-sensitive anion channels in beta-cells but lowered intracellular pH and adversely affected both the bioelectrical response to d-glucose and its effect on the cytosolic concentration of Ca2+ in these cells. The lowering of cellular pH by acetazolamide, which could well be due to inhibition of carbonic anhydrase, might in turn account for inhibition of glycolysis. The perturbation of stimulus-secretion coupling in the beta-cells exposed to acetazolamide may thus involve impaired circulation in the pyruvate-malate shuttle, altered mitochondrial Ca2+ accumulation, and perturbation of Cl- fluxes, resulting in both decreased bioelectrical activity and insulin release.

Oscillatory Membrane Potential Response to Glucose in Islet β-Cells: A Comparison of Islet-Cell Electrical Activity in Mouse and Rat

In contrast to mouse, rat islet beta-cell membrane potential is reported not to oscillate in response to elevated glucose despite demonstrated oscillations in calcium and insulin secretion. We aim to clarify the electrical activity of rat islet beta-cells and characterize and compare the electrical activity of both alpha- and beta-cells in rat and mouse islets. We recorded electrical activity from alpha- and beta-cells within intact islets from both mouse and rat using the perforated whole-cell patch clamp technique. Fifty-six percent of both mouse and rat beta-cells exhibited an oscillatory response to 11.1 mm glucose. Responses to both 11.1 mm and 2.8 mm glucose were identical in the two species. Rat beta-cells exhibited incremental depolarization in a glucose concentration-dependent manner. We also demonstrated electrical activity in human islets recorded under the same conditions. In both mouse and rat alpha-cells 11 mm glucose caused hyperpolarization of the membrane potential, whereas 2.8 mm glucose produced action potential firing. No species differences were observed in the response of alpha-cells to glucose. This paper is the first to demonstrate and characterize oscillatory membrane potential fluctuations in the presence of elevated glucose in rat islet beta-cells in comparison with mouse. The findings promote the use of rat islets in future electrophysiological studies, enabling consistency between electrophysiological and insulin secretion studies. An inverse response of alpha-cell membrane potential to glucose furthers our understanding of the mechanisms underlying glucose sensitive glucagon secretion.

Ghrelin attenuates glucose-induced insulin release via inhibition of cyclic AMP productions in rat islet β-cells

Ghrelin attenuates glucose-induced insulin release via inhibition of cyclic AMP productions in rat islet β-cells

Oleic acid interacts with GPR40 to induce Ca2+signaling in rat islet β-cells: mediation by PLC and L-type Ca2+channel and link to insulin release

It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic beta-cells. Recently, it was reported that FFAs function as endogenous ligands for GPR40, a G protein-coupled receptor, to amplify glucose-stimulated insulin secretion in an insulinoma cell line and rat islets. However, signal transduction mechanisms for GPR40 in beta-cells are little known. The present study was aimed at elucidating GPR40-linked Ca(2+) signaling mechanisms in rat pancreatic beta-cells. We employed oleic acid (OA), an FFA that has a high affinity for the rat GPR40, and examined its effect on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single beta-cells by fura 2 fluorescence imaging. OA at 1-10 microM concentration-dependently increased [Ca(2+)](i) in the presence of 5.6, 8.3, and 11.2 mM, but not 2.8 mM, glucose. OA-induced [Ca(2+)](i) increases at 11.2 mM glucose were inhibited in beta-cells transfected with small interfering RNA targeted to rat GPR40 mRNA. OA-induced [Ca(2+)](i) increases were also inhibited by phospholipase C (PLC) inhibitors, U73122 and neomycin, Ca(2+)-free conditions, and an L-type Ca(2+) channel blocker, nitrendipine. Furthermore, OA increased insulin release from isolated islets at 8.3 mM glucose, and it was markedly attenuated by PLC and L-type Ca(2+) channel inhibitors. These results demonstrate that OA interacts with GPR40 to increase [Ca(2+)](i) via PLC- and L-type Ca(2+) channel-mediated pathway in rat islet beta-cells, which may be link to insulin release.

Intragranular targeting of syncollin, but not a syncollinGFP chimera, inhibits regulated insulin exocytosis in pancreatic β-cells

Several proteins play a role in the mechanism of insulin exocytosis. However, these 'exocytotic proteins' have yet to account for the regulated aspect of insulin exocytosis, and other factors are involved. In pancreatic exocrine cells, the intralumenal zymogen granule protein, syncollin, is required for efficient regulated exocytosis, but it is not known whether intragranular peptides similarly influence regulated insulin exocytosis. Here, this issue has been addressed using expression of syncollin and a syncollin-green fluorescent protein (syncollinGFP) chimera in rat islet beta-cells as experimental tools. Syncollin is not normally expressed in beta-cells but adenoviral-mediated expression of both syncollin and syncollinGFP indicated that these were specifically targeted to the lumen of beta-granules. Syncollin expression in isolated rat islets had no effect on basal insulin secretion but significantly inhibited regulated insulin secretion stimulated by glucose (16.7 mM), glucagon-like peptide-1 (GLP-1) (10 nM) and glyburide (5 microM). Consistent with specific localization of syncollin to beta-granules, constitutive secretion was unchanged by syncollin expression in rat islets. Syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production. Moreover, secretagogue-induced increases in cytosolic intracellular Ca2+, which is a prerequisite for triggering insulin exocytosis, were unaffected in syncollin-expressing islets. Therefore, syncollin was most likely acting downstream of secondary signals at the level of insulin exocytosis. Thus, syncollin expression in beta-cells has highlighted the importance of intralumenal beta-granule peptide factors playing a role in the control of insulin exocytosis. In contrast to syncollin, syncollinGFP had no effect on insulin secretion, underlining its usefulness as a 'fluorescent tag' to track beta-granule transport and exocytosis in real time.

Pertussis toxin-sensitive pathway inhibits glucose-stimulated Ca 2+ signals of rat islet β-cells by affecting L-type Ca 2+ channels and voltage-dependent K + channels

A role of pertussis toxin (PTX)-sensitive pathway in regulation of glucose-stimulated Ca 2+ signaling in rat islet β-cells was investigated by using clonidine as a selective agonist to α 2-adrenoceptors which link to the pathway. An elevation of extracellular glucose concentration from 5.5 to 22.2 mM (glucose stimulation) increased the levels of [Ca 2+] i of β-cells, and clonidine reversibly reduced the elevated levels of [Ca 2+] i. This clonidine effect was antagonized by yohimbine, and abolished in β-cells pre-treated with PTX. Clonidine showed little effect on membrane currents including those through ATP-sensitive K + channels induced by voltage ramps from −90 to −50 mV. Clonidine showed little effect on the magnitude of whole-cell currents through L-type Ca 2+ channels ( I Ca(L)), but increased the inactivation process of the currents. Clonidine increased the magnitude of the voltage-dependent K + currents ( I VK). These clonidine effects on I Ca(L) and I VK were abolished in β-cells treated with PTX or GDP-βS. These results suggest that the PTX-sensitive pathway increases I VK activity and decreases I Ca(L) activity of islet β-cells, resulting in a decrease in the levels of [Ca 2+] i elevated by depolarization-induced Ca 2+ entry. This mechanism seems responsible at least in part for well-known inhibitory action of PTX-sensitive pathway on glucose-stimulated insulin secretion from islet β-cells.

Correlation between GABA release from rat islet beta-cells and their metabolic state.

Pancreatic beta-cells express glutamate decarboxylase (GAD), which is responsible for the production and release of gamma-aminobutyric acid (GABA). Over a 24-h culture period, total GABA release by purified rat beta-cells is eightfold higher than the cellular GABA content and can thus be used as an index of cellular GAD activity. GABA release is 40% reduced by glucose (58 pmol/10(3) cells at 10 mM glucose vs. 94 pmol at 3 mM glucose, P < 0.05). This suppressive effect of glucose was not observed when glucose metabolism was blocked by mannoheptulose or 2,4-dinitrophenol; it was amplified when ATP-dependent beta-cell activities were inhibited by addition of diazoxide, verapamil, or cycloheximide or by reduction of extracellular calcium levels; it was counteracted when beta-cell functions were activated by nonmetabolized agents, such as glibenclamide, IBMX, glucagon, or glucacon-like peptide-1 (GLP-1), which are known to stimulate calcium-dependent activities, such as hormone release and calcium-dependent ATPases. These observations suggest that GABA release from beta-cells varies with the balance between ATP-producing and ATP-consuming activities in the cells. Less GABA is released in conditions of elevated glucose metabolism, and hence ATP production, but this effect is counteracted by ATP-dependent activities. The notion that increased cytoplasmic ATP levels can suppress GAD activity in beta-cells, and hence GABA production and release, is compatible with previous findings on ATP suppression of brain GAD activity.