Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).     

Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells.

Synaptotagmin (Syt) I-deficient phaeochromocytoma (PC12) cell lines show normal Ca(2+)-dependent norepinephrine (NE) release (Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R., and Takahashi, M. (1992) Science 256, 1821-1823). To identify an alternative Ca(2+) sensor, we searched for other Syt isoforms in Syt I-deficient PC12 cells and identified Syt IX, an isoform closely related to Syt I, as an abundantly expressed dense-core vesicle protein. Here we show that Syt IX is required for the Ca(2+)-dependent release of NE from PC12 cells. Antibodies directed against the C2A domain of either Syt IX or Syt I inhibited Ca(2+)-dependent NE release in permeable PC12 cells indicating that both Syt proteins function in dense-core vesicle exocytosis. Our results support the idea that Syt family proteins that co-reside on secretory vesicles may function cooperatively and redundantly as potential Ca(2+) sensors for exocytosis.     

Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-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.     

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.     

Synaptotagmin I and Ca(2+) promote half fusion more than full fusion in SNARE-mediated bilayer fusion

Synaptic membrane fusion, which is necessary for neurotransmitter release, may be mediated by SNAREs and regulated by synaptotagmin (Syt) and Ca(2+). Fusion of liposomes mediated by reconstituted SNAREs produces full fusion and hemifusion, a membrane structure in which outer leaflets are mixed but the inner leaflets remain intact. Here, using the liposome fusion assay, it is shown that Syt promoted both hemifusion and full fusion in a Ca(2+)-dependent manner. Syt.Ca(2+) increased hemifusion more than full fusion, modulating the ratio of hemifusion to full fusion. Unlike the case of neuronal SNAREs, stimulation of fusion by Syt.Ca(2+) was not seen for other SNAREs involved in trafficking in yeast, indicating that the Syt.Ca(2+) stimulation was SNARE-specific. We constructed hybrid SNAREs in which transmembrane domains were swapped between neuronal and yeast SNAREs. With these hybrid SNAREs, we demonstrated that the interaction between the SNARE motifs of neuronal proteins and Syt.Ca(2+) was required for the stimulation of fusion.     

Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli

Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium.     

Free fatty acid accumulation in secretagogue-stimulated pancreatic islets and effects of arachidonate on depolarization-induced insulin secretion

Free fatty acids in isolated pancreatic islets have been quantified by gas chromatography-mass spectrometry after stimulation with insulin secretagogues. The fuel secretagogue D-glucose has been found to induce little change in islet palmitate levels but does induce the accumulation of sufficient unesterified arachidonate by mass to achieve an increment in cellular levels of 38-75 microM. Little of this free arachidonate is released into the perifusion medium, and most remains associated with the islets. Glucose-induced hydrolysis of arachidonate from islet cell phospholipids is reflected by release of the arachidonate metabolite prostaglandin E2 (PGE2) from perifused islets. Both the depolarizing insulin secretagogue tolbutamide (which is thought to act by inducing closure of beta-cell ATP-sensitive K+ channels and the influx of extracellular Ca2+ through voltage-dependent channels) and the calcium ionophore A23187 have also been found to induce free arachidonate accumulation within and PGE2 release from islets. Surprisingly, a major fraction of glucose-induced eicosanoid release was found not to require Ca2+ influx and occurred even in Ca(2+)-free medium, in the presence of the Ca(2+)-chelating agent EGTA, and in the presence of the Ca2+ channel blockers verapamil and nifedipine. Exogenous arachidonic acid was found to amplify the insulin secretory response of perifused islets to submaximally depolarizing concentrations of KCl, and the maximally effective concentration of arachidonate was 30-40 microM. These observations suggest that glucose-induced phospholipid hydrolysis and free arachidonate accumulation in pancreatic islets are not simply epiphenomena associated with Ca2+ influx and that arachidonate accumulation may play a role in the signaling process which leads to insulin secretion.     

Depolarizing stimuli reduce Ca(2+)/calmodulin-dependent protein kinase II activity in islets of Langerhans

Elevations in intracellular Ca(2+) ([Ca(2+)](i)) initiate insulin secretion from pancreatic beta-cells, but the secretory responses become rapidly desensitised to maintained elevations in [Ca(2+)](i). We have investigated the mechanisms underlying the Ca(2+) desensitization of insulin secretion using electrically permeabilized rat islets of Langerhans. Measurements of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) enzyme activity and immunoreactivity in permeabilized islets demonstrated Ca(2+)-induced reductions in enzyme activity which could not be attributed to reductions in CaMK II immunoreactive protein. Measurements in intact islets demonstrated that the Ca(2+)-induced reduction of CaMK II activity was also operative in intact cells, suggesting that this mechanism may have pathophysiological implications for beta-cell function.     

Secretory and electrophysiological characteristics of insulin cells from gastrectomized mice: evidence for the existence of insulinotropic agents in the stomach

Mice were subjected to gastrectomy (GX) or sham operation (controls). Four to six weeks later the pancreatic islets were isolated and analysed for cAMP or alternatively incubated in a Krebs-Ringer based medium in an effort to study insulin secretion and cAMP accumulation in response to glucose or the adenylate cyclase activator forskolin. Freshly isolated islets from GX mice had higher cAMP content than islets from control mice, a difference that persisted after incubation for 1 h at a glucose concentration of 4 mmol/l. Addition of forskolin to this medium induced much greater cAMP and insulin responses in islets from GX mice than in islets from control mice. In contrast, the insulin response to high glucose (16.7 mmol/l) was much weaker in GX islets than in control islets. Glucose-induced insulin release was associated with a 2-fold rise in the cAMP content in control islets. Surprisingly no rise in cAMP was noted in GX islets incubated at high glucose. Capacitance measurements conducted on isolated insulin cells from GX mice revealed a much lower exocytotic response to a single 500 ms depolarisation (from -70 mV to zero) than in control insulin cells. Addition of cAMP to the cytosol enhanced the exocytotic response in insulin cells from control mice but not from GX mice. The depolarisation-triggered inward Ca(2+) current in insulin cells from GX mice did not differ from that in control mice, and hence the reduced exocytotic response following GX cannot be ascribed to a decreased Ca(2+) influx. Experiments involving a train of ten 500 ms depolarisations revealed that the exocytotic response was prominent in control insulin cells but modest in GX insulin cells. It seems that cAMP is capable of eliciting insulin release from insulin cells of GX mice only when cAMP is generated in a specific microdomain conceivably through the intervention of membrane-associated adenylate cyclases that can be activated by forskolin. The GX-evoked impairment of depolarisation-induced exocytosis and glucose-stimulated insulin release may reflect the lack of a gastric agent that serves to maintain an appropriate insulin response to glucose and an appropriate exocytotic response to depolarisation by raising cAMP in a special glucose-sensitive compartment possibly regulated by a soluble adenylate cyclase.     

Augmentation of basal insulin release from rat islets by preexposure to a high concentration of glucose

We have found that preexposure to an elevated concentration of glucose reversibly induces an enhancement of basal insulin release from rat pancreatic islets dependent on glucose metabolism. This basal insulin release augmented by priming was not suppressed by reduction of the intracellular ATP or Ca(2+) concentration, because even in the absence of ATP at low Ca(2+), the augmentation was not abolished from primed electrically permeabilized islets. Moreover, it was not inhibited by an alpha-adrenergic antagonist, clonidine. A threshold level of GTP is required to induce these effects, because together with adenine, mycophenolic acid, a cytosolic GTP synthesis inhibitor, completely abolished the enhancement of basal insulin release due to the glucose-induced priming without affecting the glucose-induced increment in ATP content and ATP-to-ADP ratio. In addition, a GDP analog significantly suppressed the enhanced insulin release due to priming from permeabilized islets in the absence of ATP at low Ca(2+), suggesting that the GTP-sensitive site may play a role in the augmentation of basal insulin release due to the glucose-induced priming effect.     

Antidiabetic effect of a prodrug of cysteine, L-2-oxothiazolidine-4-carboxylic acid, through CD38 dimerization and internalization.

CD38 is a bifunctional enzyme synthesizing (ADP-ribosyl cyclase) and degrading (cyclic ADP-ribose (cADPR) hydrolase) cADPR, a potent Ca(2+) mobilizer from intracellular pools. CD38 internalization has been proposed as a mechanism by which the ectoenzyme produced intracellular cADPR, and thiol compounds have been shown to induce the internalization of CD38. Here, we show that the disulfide bond between Cys-119 and Cys-201 in CD38 may be involved in CD38 dimerization and internalization. We tested the effect of a reducing agent, l-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, on CD38 internalization in pancreatic islets. OTC enhanced insulin release from isolated islets as well as CD38 internalization and cytoplasmic Ca(2+) level. Furthermore, islet cells treated with antisense CD38 oligonucleotide showed inhibition of OTC-induced insulin secretion. Intake of OTC in db/db mice ameliorated glucose tolerance, insulin secretion, and morphology of islets when compared with control mice. These data indicate that OTC improves glucose tolerance by enhancing insulin secretion via CD38/cADPR/Ca(2+) signaling machinery. Thus, OTC may represent a novel class of antidiabetic drug.     

Differential distribution of synaptotagmin-1, -4, -7, and -9 in rat adrenal chromaffin cells

Neurons and certain kinds of endocrine cells, such as adrenal chromaffin cells, have large dense-core vesicles (LDCVs) and synaptic vesicles or synaptic-like microvesicles (SLMVs). These secretory vesicles exhibit differences in Ca(2+) sensitivity and contain diverse signaling substances. The present work was undertaken to identify the synaptotagmin (Syt) isoforms present in secretory vesicles. Fractionation analysis of lysates of the bovine adrenal medulla and immunocytochemistry in rat chromaffin cells indicated that Syt 1 was localized in LDCVs and SLMVs, whereas Syt 7 was the predominant isoform present in LDCVs. In contrast to PC12 cells and the pancreatic β cell line INS-1, Syt 9 was not immunodetected in LDCVs in rat chromaffin cells. Double-staining revealed that Syt 9-like immunoreactivity was nearly identical with fluorescent thapsigargin binding, suggesting the presence of Syt 9 in the endoplasmic reticulum (ER).The exogenous expression of Syt 1-GFP in INS-1 cells, which had a negligible level of endogenous Syt 1, resulted in an increase in the amount of Syt 9 in the ER, suggesting that Syt 9 competes with Syt 1 for trafficking from the ER to the Golgi complex. We conclude that LDCVs mainly contain Syt 7, whereas SLMVs contain Syt 1, but not Syt 7, in rat and bovine chromaffin cells.     

TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion

There are eight thermosensitive TRP (transient receptor potential) channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Here, we demonstrate that TRPM2 can be activated by exposure to warm temperatures (>35 degrees C) apparently via direct heat-evoked channel gating. beta-NAD(+)- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We also show that, even though cyclic ADP-ribose (cADPR) does not activate TRPM2 at 25 degrees C, co-application of heat and intracellular cADPR dramatically potentiates TRPM2 activity. Heat and cADPR evoke similar responses in rat insulinoma RIN-5F cells, which express TRPM2 endogenously. In pancreatic islets, TRPM2 is coexpressed with insulin, and mild heating of these cells evokes increases in both cytosolic Ca(2+) and insulin release, which is K(ATP) channel-independent and protein kinase A-mediated. Heat-evoked responses in both RIN-5F cells and pancreatic islets are significantly diminished by treatment with TRPM2-specific siRNA. These results identify TRPM2 as a potential molecular target for cADPR, and suggest that TRPM2 regulates Ca(2+) entry into pancreatic beta-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion.     

Cell swelling-induced signaling for insulin secretion bypasses steps involving G proteins and PLA2 and is N-ethylmaleimide insensitive.

BACKGROUND: This study was undertaken to examine putative mechanisms of calcium independent signal transduction pathway of cell swelling-induced insulin secretion. METHODS: The role of phospholipase A(2), G proteins, and soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) in insulin secretion induced by 30% hypotonic medium was studied using isolated rat pancreatic islets. RESULTS: In contrast to glucose stimulation, osmotically induced insulin secretion from pancreatic islets was not inhibited by 10 micromol/l bromoenol lactone, an iPLA(2) (Ca(2+) independent phospholipase) inhibitor. Similarly, preincubation of islets for 20 hours with 25 microg/ml mycophenolic acid to inhibit GTP synthesis fully abolished glucose-induced insulin secretion but was without effect on hypotonicity stimulated insulin release. Glucose-induced insulin secretion was prevented by preincubation with 20 nmol/l tetanus toxin (TeTx), a metalloprotease inactivating soluble SNARE. Cell swelling-induced insulin secretion was inhibited by TeTx in the presence of calcium ions but not in calcium depleted medium. The presence of N-ethylmaleimide (NEM, 5 mmol/l, another inhibitor of SNARE proteins) in the medium resulted in high basal insulin secretion and lacking response to glucose stimulation. In contrast, high basal insulin secretion from NEM treated islets further increased after hypotonic stimulation. CONCLUSION: G proteins and iPLA(2) - putative mediators of Ca(2+) independent signaling pathway participate in glucose but not in hypotonicity-induced insulin secretion. Hypotonicity-induced insulin secretion is sensitive to clostridial neurotoxin TeTx but is resistant to NEM.     

Secretory phospholipase A2 is released from pancreatic beta-cells and stimulates insulin secretion via inhibition of ATP-dependent K+ channels

The release of sPLA(2) from single mouse pancreatic beta-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of beta-cells with agents that increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) induced a rapid release of sPLA(2) to the extracellular medium. Exogenous sPLA(2) strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA(2) was mediated via inhibition of ATP-dependent K(+) channels and an increase in [Ca(2+)](i). Measurements of cell capacitance in single beta-cells revealed that sPLA(2) did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA(2) is operational in pancreatic beta-cells and point to this enzyme as an autocrine regulator of insulin secretion.     

R-type Ca(2+)-channel-evoked CICR regulates glucose-induced somatostatin secretion

Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing beta-cells and glucagon-secreting alpha-cells, the islets contain somatostatin-releasing delta-cells. Somatostatin is a powerful inhibitor of insulin and glucagon secretion. It is normally secreted in response to glucose and there is evidence suggesting its release becomes perturbed in diabetes. Little is known about the control of somatostatin release. Closure of ATP-regulated K(+)-channels (K(ATP)-channels) and a depolarization-evoked increase in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (>or=10 mM) is unaffected by the K(ATP)-channel activator diazoxide and proceeds normally in K(ATP)-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca(2+)-induced Ca(2+)-release (CICR). This constitutes a novel mechanism for K(ATP)-channel-independent metabolic control of pancreatic hormone secretion.     

Agouti signaling protein stimulates islet amyloid polypeptide (amylin) secretion in pancreatic beta-cells

Ectopic overexpression of the murine agouti gene results in yellow coat color, obesity, hyperinsulinemia, and type II diabetes. We have shown the human homologue of agouti (agouti signaling protein; ASP) to regulate human adipocyte metabolism and lipid storage via a Ca(2+)-dependent mechanism. We have also demonstrated agouti expression in human pancreas, and that ASP stimulates insulin release via a similar Ca(2+)-dependent mechanism. Plasma amylin is also elevated in agouti mutant mice. Amylin is cosecreted with insulin from beta-cells, and overexpression of human amylin in beta-cells in yellow agouti mutant mice resulted in accelerated pancreatic amyloid deposition, severely impaired beta-cell function, and a diabetic phenotype. We report here that ASP stimulates amylin release in both the HIT-T15 beta-cell line and human pancreatic islets in the presence of a wide range of glucose concentrations (0-16.7 mmol/L), similar to its effect on insulin release; this effect was blocked by 30 mumol/L nitrendipine, confirming a Ca(2+)-dependent mechanism. Accordingly, ASP stimulation of amylin release may serve as a compensatory system to regulate blood glucose in yellow agouti mutants.     

Effect of annexin I on insulin secretion through surface binding sites in rat pancreatic islets

This study investigates the effect of extracellular annexin I (Anx I) on regulating insulin secretion in isolated rat pancreatic islets. Results show that Anx I stimulates insulin release in pancreatic islets regardless of the presence or absence of extracellular Ca2+. In particular, confocal microscopy shows that Anx I binds to the surface of islet cells in the absence of extracellular Ca2+. However, insulin secretion through Anx I significantly decreases in trypsin-treated islets. Likewise, there is minimal binding of Anx I to the surface of trypsin-treated islets. Anti-Anx I polyclonal antibody also inhibits the stimulating effect of Anx I on insulin secretion. These results indicate that Anx I is capable of binding to the cell surface receptor, in order to regulate the stimulation of insulin release in rat pancreatic islets.