Insulin Secretion In HIT-T15 Cells Research Articles

Effect of protein kinase C on glucose-mediated insulin secretion in HIT-T15 cells.

To clarify the regulation of protein kinase C on glucose-mediated insulin secretion. We examined the effect of protein kinase C on the cytosolic free Ca(2+) concentration ([Ca(2+)]i) and the activity of Ca(2+)-activated K(+) channels (K(Ca)-channel) in the insulinoma cell line, HIT-T15. Glucose at a concentration of 10 mmol/L increased the secretion of insulin. This increase was partly inhibited by 1 nmol/L staurosporine, a protein kinase C inhibitor. Staurosporine (1 nmol/L) also attenuated the glucose-induced elevations in [Ca(2+)]i. On the contrary, glibenclamide (100 nmol/L) specifically blocked ATP-sensitive K(+) channels, and increased both [Ca(2+)]i and insulin secretion, but staurosporine had no effect on them. Patch clamp studies showed that 10 mmol/L glucose almost completely blocked K(Ca) channel activity, an effect that was reversed by 1 nmol/L staurosporine. Phorbol 12-myristate 13-acetate (1 mmol/L), a protein kinase C activator, also decreased K(Ca) channel activity. These results indicate that the activation of protein kinase C is involved in the glucose-induced release of insulin by modulating K(+) channel function in HIT-T15 cells.

Caribbean maitotoxin elevates [Ca2+]iand activates non-selective cation channels in HIT-T15 cells

To investigate the cytotoxic mechanism of caribbean maitotoxin (MTX-C) in mammalian cells. We used whole-cell patch-clamp techniques and fluorescence calcium imaging to determine the cellular toxic mechanisms of MTX-C in insulin secreting HIT-T15 cells, which is a system where the effects of MTX have been observed. HIT-T15 cells stably express L-type calcium current, making it a suitable model for this study. Using the fluorescence calcium indicator Indo-1 AM, we found that there is a profound increase in HIT-T15 intracellular free calcium 3 min after application of 200 nmol/L MTX-C. About 3 min after perfusion of MTX-C, a gradual increase in free calcium concentration was observed. This elevation was sustained throughout the entire recording period. Application of MTX-C did not elicit the L-type calcium current, but large cationic currents appeared after applying MTX-C to the extracellular solution. The current-voltage relationship of the cation current is approximately linear within the voltage range from -60 to 50 mV, but flattened at voltages at -80 and -100 mV. These results indicate that MTX-C induces a non-voltage activated, inward current under normal physiological conditions, which by itself or through a secondary mechanism results in a large amount of cationic influx. The biophysical mechanism of MTX-C is different to its isoform, pacific maitotoxin (MTX-P), when the extracellular calcium is removed. We conclude that MTX-C causes the opening of non-selective, non-voltage-activated ion channels, which elevates level of intracellular calcium concentration and leads to cellular toxicities.

Alloxan에 의한 HIT-T15 세포의 산화적 손상에 대한 매실(Prunus mume Sieb. et Zucc.) 주정추출물의 세포보호효과

본 연구는 췌장베타세포인 HIT-T15 세포를 이용하여 매실주정추출물(PME)의 alloxan에 의한 산화스트레스로부터의 세포보호, 인슐린 분비능 및 항산화 효소 활성을 평가하였다. PME는 alloxan에 의해 유발된 산화스트레스로부터 세포를 보호하여 세포생존율을 증가시켰다. PME는 세포막 손상지표인 LDH 방출을 억제하였고 <TEX>$NAD^+$</TEX>/NADH ratio를 유의적으로 증가시켜 세포사멸이 억제되어짐을 확인하였다. 또한 alloxan 단독처리군에 비해 250 <TEX>${\mu}g$</TEX>/ml PME 처리군에서 인슐린 분비량이 유의성 있게 증가하였다. Alloxan과 PME를 동시에 처리하여 HIT-T15세포의 항산화효소 활성을 측정했을 때 산화스트레스에 의해 감소되었던 항산화효소 활성이 PME에 의해 보호되는 효과를 확인하였다. 이상의 연구결과로부터 PME는 세포괴사 및 DNA fragmentation을 억제하고 세포내 항산화효소 활성을 증가시켜 alloxan에 의해 유발된 산화스트레스로부터 췌장베타세포를 보호하고 이에 따른 인슐린 분비능 조절 효과가 있는 것으로 생각된다. The present study was designed to examine the potential antidiabetic and antioxidant effect of ethanol extract from <TEX>$Prunus$</TEX> <TEX>$mume$</TEX> fruit (PME) against alloxan-induced oxidative stress in pancreatic <TEX>${\beta}$</TEX>-cells, HIT-T15. To evaluate the antidiabetic effect of PME, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliu bromide (MTT) cell proliferation assay, lactate dehydrogenase (LDH) release assay, <TEX>$NAD^+$</TEX>/NADH ratio and insulin secretion were assessed. We also measured its antioxidant effect against alloxan-induced oxidative stress in the cells by assessing the levels of the antioxidant enzymes including superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx). The results of this analysis showed that alloxan significantly decreased cell viability, increased LDH leakage, and lowered <TEX>$NAD^+$</TEX> /NADH ratio and insulin secretion in HIT-T15 cells. However, PME significantly increased the viability of alloxan-treated cells and lowered LDH leakage. The intracellular <TEX>$NAD^+$</TEX> /NADH ratio and insulin secretion were also increased by 1.5~1.9-fold and 1.4-fold, respectively, after treatment with the PME. The HIT-T15 cells treated with alloxan showed significant decreases in the activities of antioxidant enzymes, while PME significantly elevated the levels of antioxidant enzymes. Based on these results, we suggest that PME could have a protective effect against the cytotoxicity and dysfunction of pancreatic <TEX>${\beta}$</TEX>-cells in the presence of alloxan-induced oxidative stress.

Alloxan에 의한 HIT-T15 세포 손상에 대한 쑥갓주정추출물의 세포보호효과

The objective of the present study was to evaluate the potential antidiabetic and antioxidant effect of the ethanol extract from Chrysanthemum cornarium L. var. spatiosum(CSE) against alloxan-induced oxidative stress in pancreatic <TEX>${\beta}$</TEX>-cells, HIT-T15. In this study, the antidiabetic effect of CSE was examined using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliu bromide(MTT) cell proliferation assay, lactate dehydrogenase(LDH) release assay, <TEX>$NAD^+$</TEX>/NADH ratio and insulin secretion. To further investigate whether CSE is involved in the antioxidant activity of alloxan-damaged HIT-T15 cells, its antioxidant effect against alloxan-induced oxidative stress was measured in HIT-T15 cells by determining the levels of antioxidant enzymes including superoxide dismutase(SOD), glutathione S-transferase(GST), glutathione reductase(GR) and glutathione peroxidase(GPx). The results of this analysis showed that alloxan significantly decreased cell viability, increased LDH leakage, and lowered <TEX>$NAD^+$</TEX>/NADH ratio and insulin secretion in HIT-T15 cells. However, CSE significantly increased the viability of alloxan-treated cells and lowered LDH leakage. The intracellular NAD+/NADH ratio and insulin secretion were also significantly increased by 1.7-fold and 1.3-fold, respectively, after treatment with 100 <TEX>${\mu}g/m{\ell}$</TEX> CSE. The HIT-T15 cells treated with alloxan showed significant decreases in the activities of antioxidant enzymes, while CSE significantly elevated the levels of antioxidant enzymes. These findings suggest that CSE could have a protective effect against cytotoxicity and dysfunction of pancreatic cells in the presence of alloxan-induced oxidative stress.

Binding of activating transcription factor 6 to the A5/Core of the rat insulin II gene promoter does not mediate its transcriptional repression

Pancreatic β-cells have a well-developed endoplasmic reticulum due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. It has been previously reported that overexpression of activating transcription factor 6 (ATF6) reduces insulin gene expression in part via upregulation of small heterodimer partner. In this study, we investigated whether ATF6 directly binds to the insulin gene promoter, and whether its direct binding represses insulin gene promoter activity. A bioinformatics analysis identified a putative ATF6 binding site in the A5/Core region of the rat insulin II gene promoter. Direct binding of ATF6 was confirmed using several approaches. Electrophoretic mobility shift assays in nuclear extracts from MCF7 cells, isolated rat islets and insulin-secreting HIT-T15 cells showed ATF6 binding to the native A5/Core of the rat insulin II gene promoter. Antibody-mediated supershift analyses revealed the presence of both ATF6 isoforms, ATF6α and ATF6β, in the complex. Chromatin immunoprecipitation assays confirmed the binding of ATF6α and ATF6β to a region encompassing the A5/Core of the rat insulin II gene promoter in isolated rat islets. Overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibited the activity of an insulin promoter-reporter by 50%. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity.

Insulin releasing and alpha-glucosidase inhibitory activity of ethyl acetate fraction of Acorus calamus in vitro and in vivo

Ethnopharmacological relevance The radix of Acorus calamus L. (AC) is widely used in the therapy of diabetes in traditional folk medicine of America and Indonesia, and we previously reported the insulin sensitizing activity of the ethyl acetate fraction of AC (ACE). Aim of the study To investigate the insulin releasing and alpha-glucosidase inhibitory activity of ACE in vitro and in vivo. Materials and methods Insulin releasing and alpha-glucosidase inhibitory effects of different fractions from AC were detected in vitro using HIT-T15 cell line and alpha-glucosidase enzyme. Furthermore, effects of ACE orally on serum glucose were detected in fasted and glucose/amylum challenged normal mice. Results AC and ACE increased insulin secretion in HIT-T15 cells as gliclazide did. As in vivo results, ACE (400 and 800 mg/kg) significantly decreased fasting serum glucose, and suppressed the increase of blood glucose levels after 2 g/kg glucose loading in normal mice. In addition, ACE as a mixed-type inhibitor inhibited alpha-glucosidase activity in vitro with an IC 50 of 0.41 μg/ml, and 100 mg/kg of it clearly reduced the increase of blood glucose levels after 5 g/kg amylum loading in normal mice. Conclusions Apart from its insulin sensitizing effect, ACE may have hypoglycemic effects via mechanisms of insulin releasing and alpha-glucosidase inhibition, and thus improves postprandial hyperglycemia and cardiovascular complications.

Phosphatase inhibitors induce defective hormone secretion in insulin-secreting cells and entry into apoptosis.

A long-term (> or =24 h) exposure of insulin-secreting HIT T15 cells to the phosphatase inhibitor, okadaic acid (OA), at concentrations inhibiting serine/threonine phosphatases 1 (PP1) and 2A (PP2A) reduced proliferation and insulin secretion. The reduced proliferation was related to the induction of apoptosis as evidenced by morphological criteria and the occurrence of internucleosomal DNA fragmentation after 15 h in 50 nM OA. The compromised insulin secretion was not simply a consequence of a lowered hormone content and cell growth, but comprised also a complete suppression of secretion stimulated by K+ depolarisation and forskolin. K+ depolarisation of HIT cells cultured for 24 h in 50 nM OA resulted in a nearly unimpaired influx of Ca2+, but did not induce secretion. These observations suggest that the secretory defect may be localised distal to Ca2+ influx in stimulus secretion coupling of insulin-secreting cells.

Dual Effect of ATP on Glucose-Induced Insulin Secretion in HIT-T15 Cells

: The study investigated the dual effect of purinergic nucleotides on the secretion of insulin from pancreatic beta cells. : The level of insulin secretion in HIT-T15 cells of static incubation was measured using a radioimmunoassay. : The adenine nucleotides reduced the level of glucose-induced insulin secretion in a concentration-dependent manner, and the relative potency order (IC50; muM) was BzATP (6.9) > ATP (20.4) >/= alpha, beta-methylene ATP (23.3) >/= 2-methylthio-ATP (24.9). Suramin and PPADS (200 muM), which are blockers of the purinergic receptors, had a little influence on the activity of ATP. However, the inhibitory effect of ATP was reversed by preincubation with oxidized ATP (200 muM), which is a P2X7 antagonist. The level of insulin secretion in these preincubated cells exposed to the purinergic nucleotides increased in the following order: ATP > alpha, beta-methylene ATP >/= 2-methylthio-ATP. A pretreatment with foskolin and PDBu (100 nM) potentiated the increasing effect of ATP on insulin secretion. The Western blotting showed the expression of P2X7 and P2Y11 receptors. : Purinergic stimulation has inhibitory activity on glucose-dependent insulin secretion through the activation of the P2X7 receptor, whereas it has enhancing effect through the activation of the P2Y11 receptor in HIT-T15 cells.

An Autocrine Insulin Feedback Loop Maintains Pancreatic β-Cell 3-Phosphorylated Inositol Lipids

Phosphatidylinositol 3-kinases (PI3Ks) have a central role in pancreatic beta-cell function. Downstream events include the regulation of K(ATP) channel activity, insulin secretion, gene transcription, and cell survival. Fewer data are available on the 3-phosphorylated inositol lipids (3-PIs) that are the primary products of these kinases. We characterized these PI3K products in insulin-secreting HIT T15 cells and were able to demonstrate, for the first time the presence of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. We then showed that glucose can significantly increase PtdIns(3,4,5)P(3), PtdIns(3,4)P(2), and notably PtdIns(3,5)P(2). We investigated the mechanism(s) whereby these molecules are generated under both basal and glucose-stimulated conditions. We postulated that insulin exocytosis could drive the rises in 3-PIs. In our experimental system, we could detect a rise in insulin secretion within 1 min of glucose stimulation, thus allowing the possibility that early rises in 3-PIs are regulated by secreted insulin. This was confirmed because blockade of the beta-cell insulin receptor completely abrogated the glucose-mediated increase of all three lipids, driving their concentrations below basal levels. Using primary pancreatic islets and either blockade of the insulin receptor or antibodies to insulin, we verified that basal insulin secretion is responsible for the maintenance of 3-PIs. Therefore, autocrine insulin signaling, a feature compromised in diabetes, is essential to up-regulate both basal and glucose-stimulated levels of a vital family of second messengers that preserve and drive pancreatic beta-cell function.

Methylmercury Induces Pancreatic β-Cell Apoptosis and Dysfunction

Mercury is a well-known toxic metal, which induces oxidative stress. Pancreatic beta-cells are vulnerable to oxidative stress. The pathophysiological effect of mercury on the function of pancreatic beta-cells remains unclear. The present study was designed to investigate the effects of methylmercury (MeHg)-induced oxidative stress on the cell viability and function of pancreatic beta-cells. The number of viable cells was reduced 24 h after MeHg treatment in a dose-dependent manner with a range from 1 to 20 microM. 2',7'-Dichlorofluorescein fluorescence as an indicator of reactive oxygen species (ROS) formation after exposure of HIT-T15 cells or isolated mouse pancreatic islets to MeHg significantly increased ROS levels. MeHg could also suppress insulin secretion in HIT-T15 cells and isolated mouse pancreatic islets. After 24 h of exposure to MeHg, HIT-T15 cells had a significant increase in mercury levels with a dose-dependent manner. Moreover, MeHg displayed several features of cell apoptosis including an increase of the sub-G1 population and annexin-V binding. Treatment of HIT-T15 cells with MeHg resulted in disruption of the mitochondrial membrane potential and release of cytochrome c from the mitochondria to the cytosol and activation of caspase-3. Antioxidant N-acetylcysteine effectively reversed the MeHg-induced cellular responses. Altogether, our data clearly indicate that MeHg-induced oxidative stress causes pancreatic beta-cell apoptosis and dysfunction.

The effect of hyaluronic acid on insulin secretion in HIT-T15 cells through the enhancement of gap-junctional intercellular communications

The transplantation of bioartificial pancreas has the potential to restore endogenous insulin secretion in type I diabetes. The bioartificial pancreas is constructed in vitro from cells and a support matrix. Hyaluronic acid (HA) is an extremely ubiquitous polysaccharide of extracellular matrix in the body and plays various biological roles. It has been suggested that high molecular weight (HMW) HA increases in the function of gap-junctional intercellular communications (GJIC) and the expression of connexin-43 (Cx43). To determine whether the function of pancreatic β-cells is affected by gap junctions after HMW HA-treatment, we exposed HIT-T15, a clonal pancreatic β-cell line, in various concentrations of HA for 24 h, and then detected the insulin secretion and content, using an insulin assay kit by ELISA technique. The cellular functions of GJIC were assayed by dye-transfer method using the dye solution of Lucifer yellow. HA-treatment resulted in the enhancement of GJIC function, the increase of insulin release and insulin content. The results obtained in this study suggest that HA-coating increases the insulin secretion of HIT-T15 cells by the enhancement of Cx43-mediated GJIC. The results give useful information on design biocompatibility of HA when is used as a biomaterial for bioartificial pancreas.

β-Cell toxicity of ATP-sensitive K + channel-blocking insulin secretagogues

A prolonged exposure of isolated pancreatic islets to insulin secretagogues, the imidazolines phentolamine, alinidine and idazoxan (100 μM each), the sulfonylurea tolbutamide (500 μM), or the alkaloid quinine (100 μM) resulted in morphological damage of 4–18% of β-cells compared to less than 2% in controls. Thus, the question arose whether K ATP channel-blocking insulin secretagogues are β-cell toxic as has already been suggested for sulfonylureas. The concentration- and time-dependency of the secretagogue-associated toxicity was documented by viability assays in insulin-secreting HIT T15 cells. Treatment for 24 h with idazoxan reduced MTT conversion by 50% at 100 μM and by 98% at 1000 μM. Phentolamine and quinine reduced viability comparably at 1000 μM, but were less toxic at 100 μM. On the other hand, the imidazoline alinidine and the sulfonylurea tolbutamide were only moderately toxic (less than 40% viability loss at 1000 μM). The imidazoline efaroxan appeared even to be non-toxic. Apoptotic DNA fragmentation and DEVD-caspase activation was observed at 100 μM of idazoxan and phentolamine, whereas at 1000 μM signs of necrosis predominated. Alinidine, tolbutamide and quinine treatment did not increase markers of apoptotic cell death. Blocking Ca 2+ influx by D600 did not diminish secretagogue-associated toxicity. Electron microscopy confirmed the validity of these observations for β-cells in intact mouse islets. In summary, β-cell toxicity of the tested insulin secretagogues varied widely and did not depend on a prolonged Ca 2+ influx via L-type Ca 2+ channels. Thus, secretagogue-mediated closure of K ATP channels is apparently not per se β-cell toxic.

GTP-binding protein-independent potentiation by mastoparan of IL-1beta-induced nitric oxide release from insulin-secreting HIT-T15 cells.

Our recent data implicated small molecular weight G-proteins (e.g., H-Ras) in interleukin 1beta (IL 1beta)-induced metabolic dysfunction and apoptotic demise of the islet beta cell (Tannous et al., Biochem Pharmacol 2001; 62:1459-1468, Kowluru and Morgan, Biochem Pharmacol, 2002; 63:1027-1035, Chen et al. Biochem Pharmacol, 2003; 66:1681-1694). Recently, we have shown that mastoparan, a tetradecapeptide from wasp venom, has been shown to directly activate islet endogenous G-proteins and regulate islet function (Amin et al., Endocrinology 2003; 144: 4508-4518). Herein, we investigated potential contributory roles, if any, of mastoparan (Mas)-sensitive G-proteins in IL-induced nitric oxide (NO) release from insulin-secreting HIT-T15 cells. While, ineffective by itself, Mas significantly potentiated IL-induced NO release from HIT-T15 cells. Interestingly, Mas-17, an inactive analog of Mas, also potentiated IL-induced NO release, suggesting that the potentiating effect of Mas may not involve activation of specific G-proteins. Such potentiating effects on IL-induced NO release were also demonstrable in the presence of another polycationic compound, melittin. Together, these findings suggest that Mas-induced potentiation of IL-induced NO release may in part be due to its amphiphilic and polycationic nature. These data also warrant caution in the use of Mas to study its regulation of cellular function without the use of an appropriate negative control, such as Mas-17.

Muscarinic Agonists Activate Ca 2+ Store-operated and -independent Ionic Currents in Insulin-secreting HIT-T15 Cells and Mouse Pancreatic �-Cells

The neurotransmitter acetylcholine, a muscarinic receptor agonist, augments glucose-induced insulin secretion from pancreatic beta-cells by depolarizing the membrane to enhance voltage-gated Ca(2+) influx. To clarify the electrical events involved in this process, we measured ionic currents from a clonal beta-cell line (HIT-T15) and mouse pancreatic beta-cells. In whole-cell recordings, the muscarinic agonist carbachol (CCh) dose-dependently and reversibly activated a voltage-independent, nonselective current (whole-cell conductance 24 pS/pF, reversal potential of approximately -15 mV). The current, which we refer to as I(musc), was blocked by atropine, a muscarinic receptor antagonist, and SKF 96365, a nonspecific ion channel blocker. The magnitude of the current decreased by 52% when extracellular Na(+) was removed, but was not affected by changes in extracellular Ca(2+), confirming that I(musc) is a nonselective current. To determine if I(musc) activates following release of Ca(2+) from an intracellular store, we blocked intracellular IP(3) receptors with heparin. Carbachol still activated a current in the presence of heparin, demonstrating the presence of a Ca(2+) store-independent, muscarinic agonist-activated ionic current in HIT cells. However, the store-independent current was smaller and had a more positive reversal potential (approximately 0 mV) than the current activated by CCh under control conditions. This result indicates that heparin had blocked a component of I(musc), which likely activates following release of stored Ca(2+). Depleting IP(3)-sensitive calcium stores with thapsigargin also activated a non-selective, SKF 96365-blockable current in HIT cells. The properties of this putative store-operated current were similar to the component of I(musc) that was blocked by heparin, being voltage-independent and reversing near -30 mV. We conclude that I(musc) consists of store-operated and store-independent components, both of which may contribute to the depolarizing action of muscarinic agonists on pancreatic beta-cells.

Role of tissue transglutaminase in GTP depletion-induced apoptosis of insulin-secreting (HIT-T15) cells

The role of tissue transglutaminase (tTG), a calcium-dependent and GTP-modulated enzyme, in apoptotic death induced by GTP depletion in islet β-cells was investigated. GTP depletion and apoptosis were induced by mycophenolic acid (MPA) in insulin-secreting HIT-T15 cells. MPA treatment increased in situ tTG activity (but not protein levels) in a dose- and time-dependent manner in parallel with the induction of apoptosis. MPA-induced increases of both tTG activity and apoptosis were entirely blocked by co-provision of guanosine but not adenosine. MPA-enhanced tTG activity could be substantially reduced by co-exposure to monodansylcadaverine or putrescine (tTG inhibitors), and largely blocked by lowering free Ca 2+ concentrations in the culture medium. However, MPA-induced cell death was either not changed or was only slightly reduced under these conditions. By contrast, a pan-caspase inhibitor (Z-VAD-FMK) entirely prevented apoptosis induced by MPA, but did not block the enhanced tTG activity, indicating that GTP depletion can induce apoptosis and activate tTG either independently or as part of a cascade of events involving caspases. Importantly, the morphological changes accompanying apoptosis could be markedly prevented by tTG inhibitors. These findings suggest that the effect of the marked increase in tTG activity in GTP depletion-induced apoptosis of insulin-secreting cells may be restricted to some terminal morphological changes.

SSTR2 mediates the somatostatin-induced increase in intracellular Ca 2+ concentration and insulin secretion in the presence of arginine vasopressin in clonal β-cell HIT-T15

The effects of somatostatin (SRIF) are mediated through the seven transmembrane receptor family that signals via Gi/Go. To date, five distinct SRIF receptors have been characterized and designated SSTR1-5. We have characterized the SRIF receptor that mediates the increase in [Ca 2+] i and insulin secretion in HIT-T15 cells (Simian virus 40-transformed Syrian hamster islets) using high affinity, subtype selective agonists for SSTR1 (L-797,591), SSTR2 (L-779,976), SSTR3 (L-796,778), SSTR4 (L-803,087), SSTR5 (L-817,818) and PRL-2903, a specific SSTR2 antagonist. In the presence of arginine vasopressin (AVP), SRIF increased [Ca 2+] i and insulin secretion. Treatment with the SSTR2 agonist L-779,976 resulted in similar responses to SRIF. In addition, L-779,976 increased both [Ca 2+] i and insulin secretion in a dose-dependent manner. Treatment with L-779,976 alone did not alter [Ca 2+] i or basal insulin secretion. In the presence of AVP, all other SRIF receptor agonists failed to increase [Ca 2+] i and insulin secretion. The effects of SRIF and L-779,976 were abolished by the SSTR2 antagonist PRL-2903. Our results suggest that the mechanism underlying SRIF-induced insulin secretion in HIT-T15 cells be mediated through the SSTR2.

Progesterone inhibits insulin secretion by a membrane delimited, non-genomic action.

In rat islets, progesterone caused a prompt concentration-dependent inhibition of glucose-stimulated insulin release with an IC50 of 10 microM at 8.4mM glucose. The inhibition was specific since both testosterone and 17beta-estradiol had no such effect. The degree of inhibition was similar in islets from male and female rats. The inhibition was not blocked in PTX-treated islets thus ruling out the Gi/Go proteins as mediators of the inhibition. Progesterone inhibited both glucose- and BayK-8644-stimulated insulin secretion in HIT-T15 cells and the IC50 vs. 10 mM glucose was also 10 microM. There was no effect on intracellular cyclic AMP concentration in the presence 0.2 and 10 mM glucose. Progesterone decreased [Ca2+]i under all conditions tested. The decrease in [Ca2+]i was due to blockade of the L-type voltage-dependent Ca2+ channels. Under Ca(2+)-free conditions, progesterone did not inhibit the stimulation of insulin release due to the combination of glucose, phorbol ester and forskolin. Thus blockade of Ca2+ entry appears to be the sole mechanism by which progesterone inhibits insulin release. As progesterone covalently linked to albumin had a similar inhibitory effect as progesterone itself, it is concluded that the steroid acts at the outer surface of the beta-cell plasma membrane. These effects would be classified as either AI or AIIb in the Mannheim classification of nongenomically initiated steroid actions.

Munc-18 Associates with Syntaxin and Serves as a Negative Regulator of Exocytosis in the Pancreatic β-Cell

The Munc-18 protein (mammalian homologue of the unc-18 gene; also called nSec1 or rbSec1) has been identified as an essential component of the synaptic vesicle fusion protein complex. The cellular and subcellular localization and functional role of Munc-18 protein in pancreatic beta-cells was investigated. Subcellular fractionation of insulin-secreting HIT-T15 cells revealed a 67-kDa protein in both cytosol and membrane fractions. Immunohistochemistry showed punctate Munc-18 immunoreactivity in the cytoplasm of rat pancreatic islet cells. Direct double-labeling immunofluorescence histochemistry combined with confocal laser microscopy revealed the presence of Munc-18 immunoreactivity in insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing cells. Syntaxin 1 immunoreactivity was detected in extracts of HIT-T15 cells, which were immunoprecipitated using Munc-18 antiserum, suggesting an intimate association of Munc-18 with syntaxin 1. Administration of Munc-18 peptide or Munc-18 antiserum to streptolysin O-permeabilized HIT-T15 cells resulted in significantly increased insulin release, but did not have any significant effect on voltage-gated Ca(2+) channel activity. The findings taken together show that the Munc-18 protein is present in insulin-secreting beta-cells and implicate Munc-18 as a negative regulator of the insulin secretory machinery via a mechanism that does not involve syntaxin-associated Ca(2+) channels.

Ca2+/calmodulin inhibition and phospholipase C-linked Ca2+ Signaling in clonal beta-cells.

Neurotransmitters and hormones, such as arginine vasopressin (AVP) and bombesin, evoke frequency-modulated repetitive Ca2+ transients in insulin-secreting HIT-T15 cells by binding to receptors linked to phospholipase C (PLC). The role of calmodulin (CaM)-dependent mechanisms in the generation of PLC-linked Ca2+ transients was investigated by use of the naphthalenesulfonamide CaM antagonists W-7 and W-13 and their dechlorinated control analogs W-5 and W-12. W-7 (10-30 microM) and W-13 (30-100 microM), but not W-5 (100 microM) and W-12 (300 microM), reversibly inhibited the AVP- and bombesin-induced Ca2+ transients. As the generation of PLC-linked Ca2+ transients requires mobilization of internal Ca2+ and Ca2+ influx through voltage-sensitive (VSCC) and -insensitive (VICC) Ca2+ channels, the effects of the W compounds on these processes were further investigated. First, W-7 dose dependently diminished K+ (45 mM)-induced Ca2+ signals (IC50, approximately 25 microM), and W-13 (100 microM) reduced the K+ (45 mM)-induced [Ca2+]i rise by about 40-60%, whereas W-5 (100 microM) and W-12 (300 microM) had no effect. In addition, W-7 (100 microM) inhibited whole cell Ca2+ currents in mouse beta-cells by about 60%. Second, pretreatment of cells (5 min) with W-7 (30 microM), but not W-5 (30 microM), inhibited agonist-induced internal Ca2+ mobilization by about 75% in Ca2+-free medium. Neither W-7 (30 microM) nor W-5 (30 microM) affected AVP (100 nM)-stimulated formation of IP3. Third, capacitative Ca2+ influx through VICC activated by thapsigargin (2 microM) in the presence of verapamil (50 microM) was inhibited by W-7 (30 microM) but not by W-5 (30 microM). As all of the W compound effects corresponded well to their reported anticalmodulin activity, a specific anticalmodulin action can be assumed. Thus, Ca2+ via activation of CaM-dependent processes could provide positive feedback on the generation of PLC-linked Ca2+ transients in HIT-T15 cells. This appears to involve CaM-dependent regulation of both mobilization of internal Ca2+ and Ca2+ influx through VSCC and VICC.

Disruption of Rab3-calmodulin interaction, but not other effector interactions, prevents Rab3 inhibition of exocytosis.

Rab GTPases regulate membrane traffic between the cellular compartments of eukaryotic cells. Rab3 is associated with secretory vesicles of neuronal and endocrine cells and controls the Ca(2+)-triggered release of neurotransmitters and hormones. To clarify the mode of action of Rab3 we generated mutants of the GTPase that do not interact efficiently with its putative effectors Rabphilin and RIM. Surprisingly, these mutants transfected in PC12 cells were still capable of inhibiting Ca(2+)-evoked secretion. Rab3 was shown previously to bind to calmodulin in a Ca(2+)-dependent manner. By replacing two arginines conserved between Rab3 isoforms, we generated a mutant with a reduced affinity for calmodulin. This mutant retained the capacity to interact with the Rab3 regulatory proteins, Rabphilin, RIM, Mss4 and RabGDI, and was correctly targeted to dense-core secretory granules. However, replacement of the two arginines abolished the ability of the GTP-bound form of Rab3 to inhibit exocytosis of catecholamine- and insulin-secreting cells. We propose that a Rab3-calmodulin complex generated by elevated Ca(2+) concentrations mediated at least some of the effects of the GTPase and limited the number of exocytotic events that occurred in response to secretory stimuli.