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 β-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 β-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 Hsp90β, 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 β-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 β-cell viability.
- MeSH
- apoptóza účinky léků MeSH
- beta-buňky cytologie metabolismus MeSH
- buněčné linie MeSH
- kyselina olejová farmakologie MeSH
- kyseliny stearové farmakologie MeSH
- lidé MeSH
- membránové proteiny biosyntéza MeSH
- regulace genové exprese účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Saturated stearic acid (SA) induces apoptosis in the human pancreatic β-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 β-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.
- MeSH
- aktivace enzymů MeSH
- apoptóza * účinky léků MeSH
- beta-buňky účinky léků metabolismus MeSH
- buněčné linie MeSH
- exprese genu MeSH
- inhibitory proteinkinas farmakologie MeSH
- kyseliny stearové farmakologie MeSH
- lidé MeSH
- MAP kinasový signální systém účinky léků MeSH
- mastné kyseliny metabolismus farmakologie MeSH
- mitogenem aktivované proteinkinasy p38 antagonisté a inhibitory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Fatty acid-induced apoptosis and ER stress of pancreatic β-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 β-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 β-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.
- MeSH
- apoptóza účinky léků MeSH
- beta-buňky cytologie metabolismus MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- fosforylace MeSH
- JNK mitogenem aktivované proteinkinasy antagonisté a inhibitory metabolismus MeSH
- kaspasa 2 chemie genetika metabolismus MeSH
- kaspasa 7 metabolismus MeSH
- kaspasa 8 metabolismus MeSH
- kaspasa 9 metabolismus MeSH
- kyseliny stearové farmakologie MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- poly(ADP-ribosa)-polymerasy metabolismus MeSH
- proteiny tepelného šoku metabolismus MeSH
- RNA interference MeSH
- signální transdukce účinky léků MeSH
- stres endoplazmatického retikula účinky léků MeSH
- transkripční faktor ATF6 metabolismus MeSH
- transkripční faktor CHOP metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
AIMS: In this study we have tested the effect of unsaturated fatty acids on the proapoptotic effects of saturated fatty acids in the human pancreatic β-cells NES2Y. RESULTS: We found that unsaturated palmitoleic and oleic acid at a concentration of 0.2 mM and higher are able to completely inhibit the proapoptotic effect of their counterpart saturated palmitic and stearic acid at a concentration of 1 mM. Apoptosis induced by stearic acid was associated with significant activation of caspase-6, -7, -9, -2 and -8, but not with significant activation of caspase-3. The activation of caspases was blocked by coincubation with oleic acid. Stearic acid treatment was not associated with a significant change in mitochondrial membrane potential, reactive oxygen species level and with cytochrome c release from mitochondria. Furthermore, stearic acid treatment was not associated with changes in p21(WAF1/CIP1), PIDD, Fas receptor and Fas ligand expression. However, we detected endoplasmic reticulum (ER) stress markers, i. e. a significant upregulation of BiP and CHOP expression as well as XBP1 mRNA splicing. These changes were inhibited by coincubation with oleic acid. CONCLUSION: Presented data indicate that oleic acid inhibits apoptosis induction by stearic acid in NES2Y cells upstream of caspase activation and ER stress induction. It does not involve an interference with the mitochondrial pathway of apoptosis induction, with p53 activation and PIDD expression as well as with Fas receptor and Fas ligand expression.
- MeSH
- aktivace enzymů MeSH
- apoptóza účinky léků MeSH
- beta-buňky cytologie účinky léků metabolismus MeSH
- cytochromy c analýza MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- endoplazmatické retikulum účinky léků metabolismus MeSH
- exprese genu účinky léků MeSH
- fyziologický stres MeSH
- kaspasy metabolismus MeSH
- kyselina olejová farmakologie MeSH
- kyseliny mastné mononenasycené farmakologie MeSH
- kyseliny stearové farmakologie MeSH
- lidé MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- messenger RNA MeSH
- mitochondrie účinky léků metabolismus MeSH
- proteiny tepelného šoku genetika metabolismus MeSH
- průtoková cytometrie MeSH
- sestřih RNA MeSH
- transformované buněčné linie MeSH
- transkripční faktor CHOP genetika metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- upregulace účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
