Targets for pollutants in rat and human pancreatic beta-cells: The effect of prolonged exposure to sub-lethal concentrations of hexachlorocyclohexane isomers on the expression of function- and survival-related proteins.

Decades after most countries banned hexachlorocyclohexane, HCH isomers still pollute the environment. Many studies described HCH as a pro-diabetic factor; nevertheless, the effect of HCH isomers on pancreatic beta-cells remains unexplored. This study investigated the effects of a one-month exposure to α-HCH, ß-HCH, and γ-HCH on protein expression in human (NES2Y) and rat (INS1E) pancreatic beta-cell lines. α-HCH and γ-HCH increased proinsulin and insulin levels in INS1E cells, while ß-HCH showed the opposite trend. α-HCH altered the expression of PKA, ATF3, and PLIN2. ß-HCH affected the expression of GLUT1, GLUT2, PKA, ATF3, p-eIF2α, ATP-CL, and PLIN2. γ-HCH altered the expression of PKA, ATF3, PLIN2, PLIN5, and IDH1. From the tested proteins, PKA, ATF3, and PLIN-2 were the most sensitive to HCH exposure and have the potential to be used as biomarkers.

Calcium channel blockers do not protect against saturated fatty acid-induced ER stress and apoptosis in human pancreatic ß-cells.

It was evidenced that saturated fatty acids (FAs) have a detrimental effect on pancreatic ß-cells function and survival, leading to endoplasmic reticulum (ER) calcium release, ER stress, and apoptosis. In the present study, we have tested the effect of three calcium influx inhibitors, i.e., diazoxide, nifedipine, and verapamil, on the apoptosis-inducing effect of saturated stearic acid (SA) in the human pancreatic ß-cell lines NES2Y and 1.1B4. We have demonstrated that the application of all three calcium influx inhibitors tested has no inhibitory effect on SA-induced ER stress and apoptosis in both tested cell lines. Moreover, these inhibitors have pro-apoptotic potential per se at higher concentrations. Interestingly, these findings are in contradiction with those obtained with rodent cell lines and islets. Thus our data obtained with human ß-cell lines suggest that the prospective usage of calcium channel blockers for prevention and therapy of type 2 diabetes mellitus, developed with the contribution of the saturated FA-induced apoptosis of ß-cells, seems rather unlikely.

Molecular Mechanisms of Apoptosis Induction and Its Regulation by Fatty Acids in Pancreatic ß-Cells.

Pancreatic ß-cell failure and death contribute significantly to the pathogenesis of type 2 diabetes. One of the main factors responsible for ß-cell dysfunction and subsequent cell death is chronic exposure to increased concentrations of FAs (fatty acids). The effect of FAs seems to depend particularly on the degree of their saturation. Saturated FAs induce apoptosis in pancreatic ß-cells, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction by saturated FAs in ß-cells are not completely elucidated. Saturated FAs induce ER stress, which in turn leads to activation of all ER stress pathways. When ER stress is severe or prolonged, apoptosis is induced. The main mediator seems to be the CHOP transcription factor. Via regulation of expression/activity of pro- and anti-apoptotic Bcl-2 family members, and potentially also through the increase in ROS production, CHOP switches on the mitochondrial pathway of apoptosis induction. ER stress signalling also possibly leads to autophagy signalling, which may activate caspase-8. Saturated FAs activate or inhibit various signalling pathways, i.e., p38 MAPK signalling, ERK signalling, ceramide signalling, Akt signalling and PKCδ signalling. This may lead to the activation of the mitochondrial pathway of apoptosis, as well. Particularly, the inhibition of the pro-survival Akt signalling seems to play an important role. This inhibition may be mediated by multiple pathways (e.g., ER stress signalling, PKCδ and ceramide) and could also consequence in autophagy signalling. Experimental evidence indicates the involvement of certain miRNAs in mechanisms of FA-induced ß-cell apoptosis, as well. In the rather rare situations when unsaturated FAs are also shown to be pro-apoptotic, the mechanisms mediating this effect in ß-cells seem to be the same as for saturated FAs. To conclude, FA-induced apoptosis rather appears to be preceded by complex cross talks of multiple signalling pathways. Some of these pathways may be regulated by decreased membrane fluidity due to saturated FA incorporation. Few data are available concerning molecular mechanisms mediating the protective effect of unsaturated FAs on the effect of saturated FAs. It seems that the main possible mechanism represents a rather inhibitory intervention into saturated FA-induced pro-apoptotic signalling than activation of some pro-survival signalling pathway(s) or metabolic interference in ß-cells. This inhibitory intervention may be due to an increase of membrane fluidity.

Markers of acute toxicity of DDT exposure in pancreatic beta-cells determined by a proteomic approach.

Many compounds have the potential to harm pancreatic beta-cells; organochlorine pollutants belong to those compounds. In this work, we aimed to find markers of acute toxicity of p,p'-DDT exposure among proteins expressed in NES2Y human pancreatic beta-cells employing 2-D electrophoresis. We exposed NES2Y cells to a high concentration (150 µM, LC96 after 72 hours) of p,p'-DDT for 24 and 30 hours and determined proteins with changed expression using 2-D electrophoresis. We have found 22 proteins that changed their expression. They included proteins involved in ER stress (GRP78, and endoplasmin), mitochondrial proteins (GRP75, ECHM, IDH3A, NDUS1, and NDUS3), proteins involved in the maintenance of the cell morphology (EFHD2, TCPA, NDRG1, and ezrin), and some other proteins (HNRPF, HNRH1, K2C8, vimentin, PBDC1, EF2, PCNA, biliverdin reductase, G3BP1, FRIL, and HSP27). The proteins we have identified may serve as indicators of p,p'-DDT toxicity in beta-cells in future studies, including long-term exposure to environmentally relevant concentrations.

Upregulation of vitamin D-binding protein is associated with changes in insulin production in pancreatic beta-cells exposed to p,p'-DDT and p,p'-DDE.

Persistent organochlorine pollutants (POPs) gradually accumulate in the human organism due to their presence in the environment. Some studies have described a correlation between the level of POPs in the human body and the incidence of diabetes, but we know little about the direct effect of POPs on pancreatic beta-cells. We exposed pancreatic beta-cells INS1E to non-lethal concentrations of p,p'-DDT (1,1'-(2,2,2-Trichloroethane-1,1-diyl)bis(4-chlorobenzene)) and p,p'-DDE (1,1'-(2,2-dichloroethene-1,1-diyl)bis(4-chlorobenzene)) for 1 month, and assessed changes in protein expression and the intracellular insulin level. 2-D electrophoresis revealed 6 proteins with changed expression in cells exposed to p,p'-DDT or p,p'-DDE. One of the detected proteins - vitamin D-binding protein (VDBP) - was upregulated in both cells exposed to p,p'-DDT, and cells exposed to p,p'-DDE. Both exposures to pollutants reduced the intracellular level of insulin mRNA, proinsulin, and insulin monomer; p,p'-DDT also slightly reduced the level of hexameric insulin. Overexpression of VDBP caused by the stable transfection of beta-cells with the gene for VDBP decreased both the proinsulin and hexameric insulin level in beta-cells similarly to the reduction detected in cells exposed to p,p'-DDT. Our data suggest that in the cells exposed to p,p'-DDT and p,p'-DDE, the increased VDBP protein level decreased the proinsulin expression in an unknown mechanism.

Hypoxia Modulates Effects of Fatty Acids on NES2Y Human Pancreatic ß-cells.

Saturated fatty acids (FAs) induce apoptosis in the human pancreatic NES2Y ß-cell line while unsaturated FAs have nearly no detrimental effect. Moreover, unsaturated FAs are capable of inhibiting the pro-apoptotic effect of saturated FAs. Hypoxia is also known to have deleterious effects on ß-cells function and viability. In the present study, we have tested the modulatory effect of hypoxia on the effect of FAs on the growth and viability of the human pancreatic NES2Y ß-cells. This study represents the first study testing hypoxia effect on effects of FAs in pancreatic ß-cells as well as in other cell types. We showed that hypoxia increased the pro-apoptotic effect of saturated stearic acid (SA). Endoplasmic reticulum stress signaling seemed to be involved while redistribution of FA transporters fatty acid translocase/cluster of differentiation 36 (FAT/CD36) and fatty acid-binding protein (FABP) do not seem to be involved in this effect. Hypoxia also strongly decreased the protective effect of unsaturated oleic acid (OA) against the pro-apoptotic effect of SA. Thus, in the presence of hypoxia, OA was unable to save SA-treated ß-cells from apoptosis induction. Hypoxia itself had only a weak detrimental effect on NES2Y cells. Our data suggest that hypoxia could represent an important factor in pancreatic ß-cell death induced and regulated by FAs and thus in the development of type 2 diabetes mellitus.

Stearate-Induced Apoptosis in Human Pancreatic ß-Cells is Associated with Changes in Membrane Protein Expression and These Changes are Inhibited by Oleate.

PURPOSE: Lipotoxicity is implicated in type 2 diabetes pathogenesis. Its molecular mechanisms are not completely understood. The aim of this study is to identify new suspect proteins involved in pancreatic ß-cell death induction by saturated fatty acids and its inhibition by unsaturated fatty acids. EXPERIMENTAL DESIGN: Employing 2DE analysis and subsequent western blot confirmation, the differences in membrane/membrane-associated protein expression in human ß-cell line NES2Y are assessed during cell death induction by stearate and its inhibition by oleate. RESULTS: Induction of apoptosis by stearate is associated with significantly increased levels of Hsp90ß, peroxiredoxin-1, and 14-3-3γ in the membrane fraction of NES2Y cells and significantly decreased levels of annexin A2, annexin A4, and reticulocalbin-2. All these changes are significantly inhibited by oleate co-application. No expression changes are detected after application of stearate together with oleate. Furthermore, the expression of reticulocalbin-2 is significantly decreased after stearate application also in the whole cell lysate. CONCLUSIONS AND CLINICAL RELEVANCE: Several membrane-associated proteins that could be related to pro- and anti-apoptotic signaling initiated by fatty acids in human pancreatic ß-cells are identified. As far as we know, annexin A4, reticulocalbin-2, and 14-3-3γ represent novel molecules related to the effect of fatty acids on ß-cell viability.

Effect of Saturated Stearic Acid on MAP Kinase and ER Stress Signaling Pathways during Apoptosis Induction in Human Pancreatic ß-Cells Is Inhibited by Unsaturated Oleic Acid.

It has been shown that saturated fatty acids (FAs) have a detrimental effect on pancreatic ß-cells function and survival, leading to apoptosis, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction and regulation by FAs in ß-cells remain unclear; however, mitogen-activated protein (MAP) kinase and endoplasmic reticulum (ER) stress signaling pathways may be involved. In this study, we tested how unsaturated oleic acid (OA) affects the effect of saturated stearic acid (SA) on the p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways as well as the ER stress signaling pathways during apoptosis induction in the human pancreatic ß-cells NES2Y. We demonstrated that OA is able to inhibit all effects of SA. OA alone has only minimal or no effects on tested signaling in NES2Y cells. The point of OA inhibitory intervention in SA-induced apoptotic signaling thus seems to be located upstream of the discussed signaling pathways.

Differing Mechanisms of Death Induction by Fluorinated Taxane SB-T-12854 in Breast Cancer Cells.

BACKGROUND/AIM: Classical taxanes are routinely used in cancer therapy. In this study, mechanisms involved in death induction by the novel fluorine-containing taxane SB-T-12854 were investigated. MATERIALS AND METHODS: We employed breast cancer SK-BR-3, MCF-7 and T47D cell lines to assess activation of individual caspases, changes in the expression of proteins of the Bcl-2 family, and the release of pro-apoptotic factors from mitochondria into the cytosol after SB-T-12854 treatment. RESULTS: Caspase-2, -8, and -9 were activated in SK-BR-3 and MCF-7 cells. Only caspase-8 was activated in T47D cells. Caspase-7 and -6 were activated in all tested cells while caspase-3 was activated only in SK-BR-3 cells. Pro-apoptotic Bad protein seems to be important for cell death induction in all tested cells. Anti-apoptotic Bcl-2 and pro-apoptotic Bim, Bok, Bid and Bik seem to be also associated with cell death induction in some of the tested cells. The mitochondrial apoptotic pathway was significantly activated in association with the release of cytochrome c and Smac from mitochondria, but only in SK-BR-3 cells, not in MCF-7 and T47D cells. CONCLUSION: Cell death induced by SB-T-12854, in the tested breast cancer cells, differs regarding activation of caspases, changes in levels of pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family and activation of the mitochondrial apoptotic pathway.

Kinase Signaling in Apoptosis Induced by Saturated Fatty Acids in Pancreatic ß-Cells.

Pancreatic ß-cell failure and death is considered to be one of the main factors responsible for type 2 diabetes. It is caused by, in addition to hyperglycemia, chronic exposure to increased concentrations of fatty acids, mainly saturated fatty acids. Molecular mechanisms of apoptosis induction by saturated fatty acids in ß-cells are not completely clear. It has been proposed that kinase signaling could be involved, particularly, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and Akt kinases and their pathways. In this review, we discuss these kinases and their signaling pathways with respect to their possible role in apoptosis induction by saturated fatty acids in pancreatic ß-cells.

p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic ß-Cells.

Saturated stearic acid (SA) induces apoptosis in the human pancreatic ß-cells NES2Y. However, the molecular mechanisms involved are unclear. We showed that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway in these cells. Therefore, we tested the role of p38 MAPK signaling pathway activation in apoptosis induction by SA in NES2Y cells. Crosstalk between p38 MAPK pathway activation and accompanying ERK pathway inhibition after SA application was also tested. The inhibition of p38 MAPK expression by siRNA silencing resulted in a decrease in MAPKAPK-2 activation after SA application, but it had no significant effect on cell viability or the level of phosphorylated ERK pathway members. The inhibition of p38 MAPK activity by the specific inhibitor SB202190 resulted in inhibition of MAPKAPK-2 activation and noticeable activation of ERK pathway members after SA treatment but in no significant effect on cell viability. p38 MAPK overexpression by plasmid transfection produced an increase in MAPKAPK-2 activation after SA exposure but no significant influence on cell viability or ERK pathway activation. The activation of p38 MAPK by the specific activator anisomycin resulted in significant activation of MAPKAPK-2. Concerning the effect on cell viability, application of the activator led to apoptosis induction similar to application of SA (PARP cleavage and caspase-7, -8, and -9 activation) and in inhibition of ERK pathway members. We demonstrated that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway and that this activation could be involved in apoptosis induction by SA in the human pancreatic ß-cells NES2Y. However, this involvement does not seem to play a key role. Crosstalk between p38 MAPK pathway activation and ERK pathway inhibition in NES2Y cells seems likely. Thus, the ERK pathway inhibition by p38 MAPK activation does not also seem to be essential for SA-induced apoptosis.

The role of individual caspases in cell death induction by taxanes in breast cancer cells.

BACKGROUND: In previous study we showed that caspase-2 plays the role of an apical caspase in cell death induction by taxanes in breast cancer cells. This study deals with the role of other caspases. We tested breast cancer cell lines SK-BR-3 (functional caspase-3) and MCF-7 (nonfunctional caspase-3). METHODS AND RESULTS: Using western blot analysis we demonstrated the activation of initiator caspase-8 and -9 as well as executioner caspase-6 and -7 in both tested cell lines after application of taxanes (paclitaxel, SB-T-1216) at death-inducing concentrations. Caspase-3 activation was also found in SK-BR-3 cells. Employing specific siRNAs after taxane application, suppression of caspase-3 expression significantly increased the number of surviving SK-BR-3 cells. Inhibition of caspase-7 expression also increased the number of surviving SK-BR-3 and MCF-7 cells. On the other hand, suppression of caspase-8 and caspase-9 expression had no significant effect on cell survival. However, caspase-9 seemed to be involved in the activation of caspase-3 and caspase-7. Caspase-3 and caspase-7 appeared to activate mutually. Furthermore, we observed a significant decrease in mitochondrial membrane potential (flow cytometric analysis) and cytochrome c release (confocal microscopy, western blot after cell fractionation) from mitochondria in SK-BR-3 cells. No such changes were observed in MCF-7 cells after taxane treatment. CONCLUSION: We conclude that the activation of apical caspase-2 results in the activation of caspase-3 and -7 without the involvement of mitochondria. Caspase-9 can be activated directly via caspase-2 or alternatively after cytochrome c release from mitochondria. Subsequently, caspase-9 activation can also lead to caspase-3 and -7 activations. Caspase-3 and caspase-7 activate mutually. It seems that there is also a parallel pathway involving mitochondria that can cooperate in taxane-induced cell death in breast cancer cells.

A new species of the Miniopterus schreibersii species complex (Chiroptera: Miniopteridae) from the Maghreb Region, North Africa.

We used an integrative approach combining cranio-dental characters, mitochondrial and nuclear data and acoustic data to show the presence in the genus Miniopterus of a cryptic species from the Maghreb region. This species was previously recognised as Miniopterus schreibersii (Kuhl, 1817). Miniopterus maghrebensis sp. nov. can be differentiated from M. schreibersii sensu stricto on the basis of cranial characters and from mitochondrial DNA and microsatellite evidence. Although slight external morphological and acoustic differences were noted between the two species, these criteria alone did not allow reliable species identification from live animals. Based on the specimens identified morphologically and/or genetically, the distribution range of M. maghrebensis sp. nov. extends from northern Morocco to south of the High Atlas Mountains and northern Tunisia. The new cryptic species is found in sympatry with M. schreibersii s.str. near coastal regions of North Africa.

Caspase-2 is involved in cell death induction by taxanes in breast cancer cells.

BACKGROUND: We studied the role of caspase-2 in apoptosis induction by taxanes (paclitaxel, novel taxane SB-T-1216) in breast cancer cells using SK-BR-3 (nonfunctional p53, functional caspase-3) and MCF-7 (functional p53, nonfunctional caspase-3) cell lines. RESULTS: Both taxanes induced apoptosis in SK-BR-3 as well as MCF-7 cells. Caspase-2 activity in SK-BR-3 cells increased approximately 15-fold within 48 h after the application of both taxanes at the death-inducing concentration (100 nM). In MCF-7 cells, caspase-2 activity increased approximately 11-fold within 60 h after the application of taxanes (300 nM). Caspase-2 activation was confirmed by decreasing levels of procaspase-2, increasing levels of cleaved caspase-2 and the cleavage of caspase-2 substrate golgin-160. The inhibition of caspase-2 expression using siRNA increased the number of surviving cells more than 2-fold in MCF-7 cells, and at least 4-fold in SK-BR-3 cells, 96 h after the application of death-inducing concentration of taxanes. The inhibition of caspase-2 expression also resulted in decreased cleavage of initiator caspases (caspase-8, caspase-9) as well as executioner caspases (caspase-3, caspase-7) in both cell lines after the application of taxanes. In control cells, caspase-2 seemed to be mainly localized in the nucleus. After the application of taxanes, it was released from the nucleus to the cytosol, due to the long-term disintegration of the nuclear envelope, in both cell lines. Taxane application led to some formation of PIDDosome complex in both cell lines within 24 h after the application. After taxane application, p21WAF1/CIP1 expression was only induced in MCF-7 cells with functional p53. However, taxane application did not result in a significant increase of PIDD expression in either SK-BR-3 or MCF-7 cells. The inhibition of RAIDD expression using siRNA did not affect the number of surviving SK-BR-3 and MCF-7 cells after taxane application at all. CONCLUSION: Caspase-2 is required, at least partially, for apoptosis induction by taxanes in tested breast cancer cells. We suggest that caspase-2 plays the role of an apical caspase in these cells. Caspase-2 seems to be activated via other mechanism than PIDDosome formation. It follows the release of caspase-2 from the nucleus to the cytosol.

Caspase-2 and JNK activated by saturated fatty acids are not involved in apoptosis induction but modulate ER stress in human pancreatic ß-cells.

BACKGROUND: Fatty acid-induced apoptosis and ER stress of pancreatic ß-cells contribute to the development of type 2 diabetes, however, the molecular mechanisms involved are unclear. AIMS: In this study we have tested the role of caspase-2 and suggested ER stress mediator JNK in saturated fatty acid-induced apoptosis of the human pancreatic ß-cells NES2Y. RESULTS: We found that stearic acid at apoptosis-inducing concentration activated ER stress signaling pathways, i.e. IRE1α, PERK and ATF6 pathways, in NES2Y cells. During stearic acid-induced apoptosis, JNK inhibition did not decrease the rate of apoptosis nor the activation of caspase-8, -9, -7 and -2 and PARP cleavage. In addition, inhibition of JNK activity did not affect CHOP expression although it did decrease the induction of BiP expression after stearic acid treatment. Caspase-2 silencing had no effect on PARP as well as caspase-8, -9 and -7 cleavage and the induction of CHOP expression, however, it also decreased the induction of BiP expression. Surprisingly, caspase-2 silencing was accompanied by increased phosphorylation of c-Jun. CONCLUSIONS: We have demonstrated that caspase-2 as well as JNK are not key players in apoptosis induction by saturated fatty acids in human pancreatic ß-cells NES2Y. However, they appear to be involved in the modulation of saturated fatty acid-induced ER stress signaling, probably by a mechanism independent of c-Jun phosphorylation.