• Publikační typ
• abstrakt z konference MeSH

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.

• MeSH
• 2D gelová elektroforéza MeSH
• beta-buňky cytologie   účinky léků   metabolismus   MeSH
• biologické markery metabolismus   MeSH
• buněčné linie MeSH
• DDT toxicita   MeSH
• hmotnostní spektrometrie MeSH
• lidé MeSH
• proteomika metody   MeSH
• regulace genové exprese účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• beta-buňky účinky léků   metabolismus   MeSH
• buněčné linie MeSH
• DDT toxicita   MeSH
• dichlordifenyldichlorethylen toxicita   MeSH
• inzulin metabolismus   MeSH
• krysa rodu rattus MeSH
• látky znečišťující životní prostředí toxicita   MeSH
• protein vázající vitamin D metabolismus   MeSH
• testy subchronické toxicity MeSH
• upregulace účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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

• Publikační typ
• abstrakt z konference MeSH

It has been shown that saturated fatty acids (FAs) have a detrimental effect on pancreatic β-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 β-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 β-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.

• MeSH
• apoptóza * MeSH
• beta-buňky cytologie   metabolismus   patologie   MeSH
• buněčné linie MeSH
• kyselina olejová metabolismus   MeSH
• kyseliny stearové metabolismus   MeSH
• lidé MeSH
• MAP kinasový signální systém * MeSH
• mitogenem aktivované proteinkinasy p38 metabolismus   MeSH
• stres endoplazmatického retikula * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Pericellular oxygen concentration represents an important factor in the regulation of cell functions, including cell differentiation, growth and mitochondrial energy metabolism. Hypoxia in adipose tissue has been associated with altered adipokine secretion profile and suggested as a possible factor in the development of type 2 diabetes. In vitro experiments provide an indispensable tool in metabolic research, however, physical laws of gas diffusion make prolonged exposure of adherent cells to desired pericellular O2 concentrations questionable. The aim of this study was to investigate the direct effect of various O2 levels (1%, 4% and 20% O2) on the proteomic profile and triglyceride accumulation in 3T3-L1 differentiated preadipocytes using gas-permeable cultureware. Following differentiation of cells under desired pericellular O2 concentrations, cell lysates were subjected to two-dimensional gel electrophoresis and protein visualization using Coomassie blue staining. Spots showing differential expression under hypoxia were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. All identified proteins were subjected to pathway analysis. We observed that protein expression of 26 spots was reproducibly affected by 4% and 1% O2 (17 upregulated and 9 downregulated). Pathway analysis showed that mitochondrial energy metabolism and triglyceride synthesis were significantly upregulated by hypoxia. In conclusion, this study demonstrated the direct effects of pericellular O2 levels on adipocyte energy metabolism and triglyceride synthesis, probably mediated through the reversed tricarboxylic acid cycle flux.

• MeSH
• 2D gelová elektroforéza MeSH
• buněčná diferenciace účinky léků   MeSH
• buňky 3T3-L1 MeSH
• citrátový cyklus účinky léků   MeSH
• down regulace účinky léků   MeSH
• kultivované buňky MeSH
• kyslík farmakologie   MeSH
• lipidy biosyntéza   MeSH
• lipogeneze účinky léků   MeSH
• myši MeSH
• permeabilita MeSH
• plyny chemie   MeSH
• počet buněk MeSH
• proteomika * MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• triglyceridy metabolismus   MeSH
• tukové buňky cytologie   účinky léků   metabolismus   MeSH
• upregulace účinky léků   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE.

• MeSH
• beta-buňky účinky léků   metabolismus   MeSH
• buněčné linie MeSH
• cytoskeletální proteiny metabolismus   MeSH
• DDT toxicita   MeSH
• dichlordifenyldichlorethylen toxicita   MeSH
• glukosa metabolismus   MeSH
• látky znečišťující životní prostředí toxicita   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Resistance of cancer cells to chemotherapeutic agents is one of the main causes of treatment failure. In order to detect proteins potentially involved in the mechanism of resistance to taxanes, we assessed differences in protein expression in MCF-7 breast cancer cells that are sensitive to paclitaxel and in the same cells with acquired resistance to paclitaxel (established in our lab). Proteins were separated using two-dimensional electrophoresis. Changes in their expression were determined and proteins with altered expression were identified using mass spectrometry. Changes in their expression were confirmed using western blot analysis. With these techniques, we found three proteins expressed differently in resistant MCF-7 cells, i.e., thyroid hormone-interacting protein 6 (TRIP6; upregulated to 650%), heat shock protein 27 (HSP27; downregulated to 50%) and cathepsin D (downregulated to 28%). Silencing of TRIP6 expression by specific siRNA leads to decreased number of grown resistant MCF-7 cells. In the present study we have pointed at some new directions in the studies of the mechanism of resistance to paclitaxel in breast cancer cells.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• antitumorózní látky fytogenní farmakologie   MeSH
• chemorezistence MeSH
• kathepsin D metabolismus   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádory prsu MeSH
• paclitaxel farmakologie   MeSH
• proteiny s doménou LIM metabolismus   MeSH
• proteiny tepelného šoku HSP27 metabolismus   MeSH
• proteom metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• apoptóza MeSH
• beta-buňky * fyziologie   MeSH
• diabetes mellitus 2. typu etiologie   MeSH
• hypoxie MeSH
• inzulin sekrece   MeSH
• Langerhansovy ostrůvky * fyziologie   patofyziologie   MeSH
• lidé MeSH
• lipidy škodlivé účinky   MeSH
• metabolismus lipidů MeSH
• nemoci slinivky břišní patofyziologie   MeSH
• nežádoucí účinky léčiv MeSH
• obezita komplikace   MeSH
• pankreas * fyziologie   patofyziologie   MeSH
• vystavení vlivu životního prostředí škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH