thapsigargin and Insulinoma

Beta cell death caused by endoplasmic reticulum (ER) stress is a key factor aggravating type 2 diabetes. Exenatide, a glucagon-like peptide (GLP)-1 receptor agonist, prevents beta cell death induced by thapsigargin, a selective inhibitor of ER calcium storage. Here, we report on our proteomic studies designed to elucidate the underlying mechanisms. We conducted comparative proteomic analyses of cellular protein profiles during thapsigargin-induced cell death in the absence and presence of exenatide in INS-1 rat insulinoma cells. Thapsigargin altered cellular proteins involved in metabolic processes and protein folding, whose alterations were variably modified by exenatide treatment. We categorized the proteins with thapsigargin initiated alterations into three groups: those whose alterations were 1) reversed by exenatide, 2) exaggerated by exenatide, and 3) unchanged by exenatide. The most significant effect of thapsigargin on INS-1 cells relevant to their apoptosis was the appearance of newly modified spots of heat shock proteins, thimet oligopeptidase and 14-3-3β, ε, and θ, and the prevention of their appearance by exenatide, suggesting that these proteins play major roles. We also found that various modifications in 14-3-3 isoforms, which precede their appearance and promote INS-1 cell death. This study provides insights into the mechanisms in ER stress-caused INS-1 cell death and its prevention by exenatide.

Topics: 14-3-3 Proteins; Animals; Cell Death; Cell Line; Endoplasmic Reticulum Stress; Exenatide; Glucagon-Like Peptide-1 Receptor; Insulinoma; Pancreatic Neoplasms; Peptides; Phosphorylation; Protein Interaction Maps; Protein Processing, Post-Translational; Proteome; Proteomics; Rats; Thapsigargin; Venoms

Diabetes manifests from a loss in functional β-cell mass, which is regulated by a dynamic balance of various cellular processes, including β-cell growth, proliferation, and death as well as secretory function. The cell cycle machinery comprised of cyclins, kinases, and inhibitors regulates proliferation. However, their involvement during β-cell stress during the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two pharmacological inducers of β-cell stress, dexamethasone and thapsigargin. We hypothesized that β-cell stress upregulates p21 to activate the apoptotic pathway and suppress cell survival signaling. To this end, p21 was adenovirally overexpressed in pancreatic rat islets and 832/13 cells. As expected, p21 overexpression resulted in decreased [(3)H]thymidine incorporation. Flow cytometry analysis in p21-transduced 832/13 cells verified lower replication, as indicated by a decreased cell population in the S phase and a block in G2/M transition. The sub-G0 cell population was higher with p21 overexpression and was attributable to apoptosis, as demonstrated by increased annexin-positive stained cells and cleaved caspase-3 protein. p21-mediated caspase-3 cleavage was inhibited by either overexpression of the antiapoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the proapoptotic proteins Bax and Bak. Therefore, an intact intrinsic apoptotic pathway is central for p21-mediated cell death. In summary, our findings indicate that β-cell apoptosis can be triggered by p21 during stress and is thus a potential target to inhibit for protection of functional β-cell mass.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dexamethasone; Diabetes Mellitus, Type 2; Enzyme Inhibitors; Gene Expression; Glucocorticoids; Insulin-Secreting Cells; Insulinoma; Mitochondria; Oncogene Protein p21(ras); Pancreatic Neoplasms; Rats; Signal Transduction; Thapsigargin; Up-Regulation

Accumulating evidence suggests that endoplasmic reticulum (ER) stress by mechanisms that include ER Ca(2+) depletion via NO-dependent down-regulation of sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) contributes to beta-cell death in type 1 diabetes. To clarify whether the molecular pathways elicited by NO and ER Ca(2+) depletion differ, we here compare the direct effects of NO, in the form of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP), with the effects of SERCA2 inhibitor thapsigargin (TG) on MAPK, nuclear factor kappaB (NFkappaB), Bcl-2 proteins, ER stress, and apoptosis. Exposure of INS-1E cells to TG or SNAP caused caspase-3 cleavage and apoptosis. Both TG and SNAP induced activation of the proapoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP). However, other classical ER stress-induced markers such as up-regulation of ER chaperone Bip and alternative splicing of the transcription factor Xbp-1 were exclusively activated by TG. TG exposure caused NFkappaB activation, as assessed by IkappaB degradation and NFkappaB DNA binding. Inhibition of NFkappaB or the Bcl-2 family member Bax pathways protected beta-cells against TG- but not SNAP-induced beta-cell death. These data suggest that NO generation and direct SERCA2 inhibition cause two quantitative and qualitative different forms of ER stress. In contrast to NO, direct ER stress induced by SERCA inhibition causes activation of ER stress signaling pathways and elicit proapoptotic signaling via NFkappaB and Bax.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 9; Cell Line, Tumor; Endoplasmic Reticulum; Insulinoma; Mitogen-Activated Protein Kinase Kinases; NF-kappa B; Nitric Oxide; Oxidative Stress; Rats; S-Nitroso-N-Acetylpenicillamine; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Thapsigargin; Transcription Factor CHOP

In RIN m5F rat insulinoma beta-cells, agonists at cannabinoid CB(1) receptors modulate insulin release. Here we investigated in these cells the effect of the activation of cannabinoid CB(1) and CB(2) receptors on intracellular Ca(2+) ([Ca(2+)](i)). The CB(1) agonist arachidonoyl-chloro-ethanolamide (ACEA), and the CB(2) agonist JWH133, elevated [Ca(2+)](i) in a way sensitive to the inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), U73122 (but not to pertussis toxin and forskolin), and independently from extracellular Ca(2+). PI-PLC-dependent Ca(2+) mobilization by ACEA was entirely accounted for by activation of inositol-1,3,4-phosphate (IP(3)) receptors on the endoplasmic reticulum (ER), whereas the effect of JWH133 was not sensitive to all tested inhibitors of IP(3) and ryanodine receptors. ACEA, but not JWH133, significantly inhibited the effect on [Ca(2+)](i) of bombesin, which acts via G(q/11)- and PI-PLC-coupled receptors in insulinoma cells. The endogenous CB(1) agonists, anandamide and N-arachidonoyldopamine, which also activate transient receptor potential vanilloid type 1 (TRPV1) receptors expressed in RIN m5F cells, elevated [Ca(2+)](i) in the presence of extracellular Ca(2+) in a way sensitive to both CB(1) and TRPV1 antagonists. These results suggest that, in RIN m5F cells, CB(1) receptors are coupled to PI-PLC-mediated mobilization of [Ca(2+)](i) and might inhibit bombesin signaling.

Topics: Animals; Arachidonic Acids; Bombesin; Calcium; Cannabinoids; Capsaicin; Cell Line, Tumor; Colforsin; Dose-Response Relationship, Drug; Dronabinol; Enzyme Inhibitors; Estrenes; Insulinoma; Neuroprotective Agents; Neurotransmitter Agents; Pertussis Toxin; Phosphoinositide Phospholipase C; Pyrrolidinones; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Sensory System Agents; Signal Transduction; Thapsigargin; TRPV Cation Channels

Beta-cell mass is regulated by a balance between beta-cell growth and beta-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the group VIA Ca2+-independent phospholipase A2 (iPLA2beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA2beta and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we address this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2beta-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and CHOP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Accumulation of ceramide during ER stress was not associated with changes in mRNA levels of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides, but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, were temporally increased in the INS-1 cells. The increases in the level of NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA2beta and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to that in V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2beta or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress ceramide generation or apoptosis in either V or OE INS-1 cells. These findings indicate that iPLA2beta activation participates in ER stress-induced INS-1 cell apoptosis by promoting ceramide generation via NSMase-catalyzed hydrolysis of sphingomyelins, raising the possibility that this pathway contributes to beta-cell apoptosis due to ER stress.

Topics: Apoptosis; Cell Line, Tumor; Ceramides; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Enzymologic; Group VI Phospholipases A2; Humans; Hydrolysis; Insulinoma; Pancreatic Neoplasms; Phosphodiesterase Inhibitors; Retroviridae; RNA, Small Interfering; Spectrometry, Mass, Electrospray Ionization; Sphingomyelin Phosphodiesterase; Sphingomyelins; Thapsigargin; Transfection

An imbalance between the rate of protein synthesis and folding capacity of the endoplasmic reticulum (ER) results in stress that has been increasingly implicated in pancreatic islet beta-cell apoptosis and diabetes. Because insulin/IGF/Akt signaling has been implicated in beta-cell survival, we sought to determine whether this pathway is involved in ER stress-induced apoptosis. Mouse insulinoma cells treated with pharmacological agents commonly used to induce ER stress exhibited apoptosis within 48 h. ER stress-induced apoptosis was inhibited by cotreatment of the cells with IGF-1. Stable cell lines were created by small-interfering RNA (siRNA) with graded reduction of insulin receptor expression, and these cells had enhanced susceptibility to ER stress-induced apoptosis and reduced levels of phospho-glycogen synthase kinase 3beta (GSK3beta). In control cells, ER stress-induced apoptosis was associated with a reduction in phospho-Akt and phospho-GSK3beta. To further assess the role of GSK3beta in ER stress-induced apoptosis, stable cell lines were created by siRNA with up to 80% reduction in GSK3beta expression. These cells were found to resist ER stress-induced apoptosis. These results illustrate that ER stress-induced apoptosis is mediated at least in part by signaling through the phosphatidylinositol 3-kinase/Akt/GSK3beta pathway and that GSK3beta represents a novel target for agents to promote beta-cell survival.

Topics: Animals; Apoptosis; Brefeldin A; Cell Line, Tumor; Endoplasmic Reticulum; Gene Expression; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Insulin; Insulin-Like Growth Factor I; Insulinoma; Islets of Langerhans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Thapsigargin; Tunicamycin

The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein. Homozygous WFS1 gene mutations cause Wolfram syndrome, characterized by insulin-deficient diabetes mellitus and optic atropy. Pancreatic beta-cells are selectively lost from the patient's islets. ER localization suggests that WFS1 protein has physiological functions in membrane trafficking, secretion, processing and/or regulation of ER calcium homeostasis. Disturbances or overloading of these functions induces ER stress responses, including apoptosis. We speculated that WFS1 protein might be involved in these ER stress responses.. Islet expression of the Wfs1 protein was analyzed immunohistochemically. Induction of Wfs1 upon ER stress was examined by Northern and Western blot analyses using three different models: human skin fibroblasts, mouse pancreatic beta-cell-derived MIN6 cells, and Akita mouse-derived Ins2 (96Y/Y) insulinoma cells. The human WFS1 gene promoter-luciferase reporter analysis was also conducted.. Islet beta-cells were the major site of Wfs1 expression. This expression was also found in delta-cells, but not in alpha-cells. WFS1 expression was transcriptionally up-regulated by ER stress-inducing chemical insults. Treatment of fibroblasts and MIN6 cells with thapsigargin or tunicamycin increased WFS1 mRNA. WFS1 protein also increased in response to thapsigargin treatment in these cells. WFS1 gene expression was also increased in Ins2 (96Y/Y) insulinoma cells. In these cells, ER stress was intrinsically induced by mutant insulin expression. The WFS1 gene promoter-luciferase reporter system revealed that the human WFS1 promoter was activated by chemically induced ER stress in MIN6 cells, and that the promoter was more active in Ins2 (96Y/Y) cells than Ins2 (wild/wild) cells.. Wfs1 expression, which is localized to beta- and delta-cells in pancreatic islets, increases in response to ER stress, suggesting a functional link between Wfs1 and ER stress.

Topics: Animals; Anti-Bacterial Agents; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Inhibitors; Fibroblasts; Gene Expression; Humans; Insulinoma; Ionophores; Islets of Langerhans; Membrane Proteins; Mice; Pancreatic Neoplasms; Promoter Regions, Genetic; Stimulation, Chemical; Thapsigargin; Transcriptional Activation; Tunicamycin; Up-Regulation

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.

Topics: Acetylcholine; Animals; Calcium; Calcium Channels; Calcium-Transporting ATPases; Carbachol; Cell Line, Tumor; Cells, Cultured; Cricetinae; Dose-Response Relationship, Drug; Heparin; Inositol 1,4,5-Trisphosphate Receptors; Insulin; Insulin Secretion; Insulinoma; Ion Channel Gating; Ion Channels; Islets of Langerhans; Mice; Muscarinic Agonists; Receptors, Cytoplasmic and Nuclear; Receptors, Muscarinic; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Apoptosis is probably the main form of beta-cell death in both type 1 diabetes mellitus (T1DM) and T2DM. In T1DM, cytokines contribute to beta-cell destruction through nuclear factor-kappaB (NF-kappaB) activation. Previous studies suggested that in T2DM high glucose and free fatty acids (FFAs) are beta-cell toxic also via NF-kappaB activation. The aims of this study were to clarify whether common mechanisms are involved in FFA- and cytokine-induced beta-cell apoptosis and determine whether TNFalpha, an adipocyte-derived cytokine, potentiates FFA toxicity through enhanced NF-kappaB activation. Apoptosis was induced in insulinoma (INS)-1E cells, rat islets, and fluorescence-activated cell sorting-purified beta-cells by oleate, palmitate, and/or cytokines (IL-1beta, interferon-gamma, TNFalpha). Palmitate and IL-1beta induced a similar percentage of apoptosis in INS-1E cells, whereas oleate was less toxic. TNFalpha did not potentiate FFA toxicity in primary beta-cells. The NF-kappaB-dependent genes inducible nitric oxide synthase and monocyte chemoattractant protein-1 were induced by IL-1beta but not by FFAs. Cytokines activated NF-kappaB in INS-1E and beta-cells, but FFAs did not. Moreover, FFAs did not enhance NF-kappaB activation by TNFalpha. Palmitate and oleate induced C/EBP homologous protein, activating transcription factor-4, and immunoglobulin heavy chain binding protein mRNAs, X-box binding protein-1 alternative splicing, and activation of the activating transcription factor-6 promoter in INS-1E cells, suggesting that FFAs trigger an endoplasmic reticulum (ER) stress response. We conclude that apoptosis is the main mode of FFA- and cytokine-induced beta-cell death but the mechanisms involved are different. Whereas cytokines induce NF-kappaB activation and ER stress (secondary to nitric oxide formation), FFAs activate an ER stress response via an NF-kappaB- and nitric oxide-independent mechanism. Our results argue against a unifying hypothesis for the mechanisms of beta-cell death in T1DM and T2DM.

Topics: Animals; Apoptosis; Carcinogens; Cell Line, Tumor; Cytokines; Drug Synergism; Endoplasmic Reticulum; Fatty Acids, Nonesterified; Flow Cytometry; Insulinoma; Interferon-gamma; Interleukin-1; Islets of Langerhans; Male; NF-kappa B; Oleic Acid; Palmitates; Pancreatic Neoplasms; Rats; Rats, Wistar; Thapsigargin; Tumor Necrosis Factor-alpha

To test whether in RINm5F rat insulinoma cells luminal acidity and the activity of a vacuolar-type proton pump are involved in calcium sequestration by intracellular calcium stores sensitive to inositol 1,4,5-trisphosphate (InsP3) we examined the effects of various proton-conducting ionophores and ammonium chloride, and of bafilomycin, a specific inhibitor of vacuolar proton pumps, on this parameter. Bafilomycin in concentrations up to 1 microM did not affect calcium sequestration by nonmitochondrial, InsP3-sensitive stores at all; 50 microM carbonylcyanide m-chlorophenylhydrazone, 50 microM monensin and 30 mM NH4Cl, which are diverse ways to dissipate transmembrane pH gradients, did not inhibit calcium sequestration. This argues against signficant involvement of internal acidity and vacuolar proton pumps in calcium sequestration by InsP3-sensitive stores in RINm5F cells. The proton-potassium-exchanging ionophore nigericin (20-100 microM), however, inhibited calcium sequestration by nonmitochondrial and InsP3-sensitive stores. This effect was dependent on the presence of potassium and could be reversed by inclusion of carbonylcyanide m-chlorophenylhydrazone or acetate in the incubation medium. Thus, the inhibitory effect of nigericin appears to be based on proton extrusion coupled to potassium influx across the membrane of calcium stores in RINm5F cells, creating an internal alkalinization of these stores. The effect of nigericin implies the continuous maintenance of an outside-to-inside potassium concentration gradient by nonmitochondrial calcium stores in RINm5F cells. This feature will be of potential interest in the identification of InsP3-sensitive calcium-storing organelles.

Topics: Ammonium Chloride; Animals; Anti-Bacterial Agents; Calcium; Cell Membrane; Cell Membrane Permeability; Endoplasmic Reticulum; Hydrogen-Ion Concentration; Inositol 1,4,5-Trisphosphate; Insulinoma; Ionophores; Islets of Langerhans; Liver; Macrolides; Nigericin; Pancreatic Neoplasms; Potassium; Proton Pump Inhibitors; Proton Pumps; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured; Vacuoles; Vanadates

This study examines the calcium store-regulated (capacitative) calcium influx pathway in the endocrine pancreatic cell line RINm5F, utilizing thapsigargin. After preincubation of the cells with the phorbol ester TPA, thapsigargin induced a sustained elevation of cytosolic calcium as well as a sustained stimulation of manganese entry, the latter being used to assess calcium influx. Thapsigargin given alone provoked a smaller and only transient elevation of cytosolic calcium and stimulation of manganese entry. The protein kinase C inhibitor staurosporine antagonized the effect of the phorbol ester. Verapamil, nifedipine, or measures to hyperpolarize the cells exerted no inhibitory action against this effect, which excludes an involvement of voltage-dependent calcium channels. In conclusion, our data shows for the first time that protein kinase C stimulation activates the capacitative calcium influx pathway of endocrine pancreatic insulin-producing cells.

Topics: Alkaloids; Animals; Biological Transport; Calcium; Cytosol; Insulin; Insulin Secretion; Insulinoma; Manganese; Pancreatic Neoplasms; Protein Kinase C; Rats; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured