ABSTRACT
This study is to investigate the effect of dexamethasone on cell apoptosis of murine MIN6 pancreatic beta-cells, and to investigate the mechanism of dexamethasone-dependent cell apoptosis. The cell apoptosis model was established by choosing the murine MIN6 pancreatic beta-cells, which was cultured in vitro and induced by dexamethasone. The morphology of the cell apoptosis was observed through fluorescence microscopic analysis after Hochest/PI staining and flow cytometric assay after Annexin-V/PI staining. The expression of caspase-3 was detected with caspase-3 activity assay kit. The expressions of Cyt-c, Bcl-2, Bax, AKT and p-AKT were observed with Western blotting. The results indicated that after exposure to dexamethasone at a concentration ranging from 50-800 nmol x L(-1) for 48 h, the percentage of cell apoptosis was significantly increased with the concentration over 100 nmol x L(-1) of dexamethasone; after exposure to dexamethasone (100 nmol x L(-1)) for 72 h, the activity of caspase-3 increased significantly; after exposure to dexamethasone at a concentration ranging from 50-800 nmol x L(-1) for 48 h, the expression of Cyt-c increased, Bcl-2 and AKT phosphorylation decreased while Bax and T-AKT remained unchanged. It could be concluded that the effect of dexamethasone on murine MIN6 pancreatic beta-cells apoptosis is significant. The mechanism of dexamethasone-dependent cell apoptosis is probably related to down regulation of the Bcl-2 expression and reduction of AKT phosphorylation.
Subject(s)
Animals , Mice , Antineoplastic Agents, Hormonal , Pharmacology , Apoptosis , Caspase 3 , Metabolism , Cell Line , Cytochromes c , Metabolism , Dexamethasone , Pharmacology , Down-Regulation , Insulin-Secreting Cells , Metabolism , Pathology , Phosphorylation , Proto-Oncogene Proteins c-akt , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Metabolism , bcl-2-Associated X Protein , MetabolismABSTRACT
<p><b>AIM</b>To investigate the chronic effect of palmitic acid (PA) on apoptosis of pancreatic islet beta-cells and the possible mechanism.</p><p><b>METHODS</b>Insulinoma cell line (MIN6 cells) were used in this study. After being incubated in PA (0.1 - 1.6 mml/L) for 24 and 48 hours, MTT method was used to evaluate the livability. After being incubated for 48 h, Hoechst-PI and Annexin-V-FTTC/PI FACS were used to estimate the apoptosis in each group, Western-blotting assay was used to estimate the protein level of p-Akt, Akt, Bax and Bcl-2.</p><p><b>RESULTS</b>Chronic PA dose-dependently (1) decreased the availability and increased the apoptosis of MIN6 cells; (2) decreased the phosphorylation of Akt and Bcl-2, but had no significant effects on Akt and Bax.</p><p><b>CONCLUSION</b>Chronic PA dose-dependently induced apoptosis of MIN6 cells, and this effect was possibly regulated by Akt/Bcl-2.</p>
Subject(s)
Animals , Mice , Apoptosis , Cell Line, Tumor , Cells, Cultured , Insulinoma , Pathology , Islets of Langerhans , Pathology , Oxidative Stress , Physiology , Palmitic Acid , Pharmacology , Proto-Oncogene Proteins , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Signal Transduction , Physiology , bcl-2-Associated X Protein , MetabolismABSTRACT
<p><b>AIM</b>To explore the possible mechanism of tert-butyl hydroperoxide (t-BHP)-induced apoptosis in murine MIN6 pancreatic beta-cells.</p><p><b>METHODS</b>MIN6 cells were cultured in vitro. Cell damage was evaluated by epifluorescence microscopy after staining with AO-EB. The percentage of cell apoptosis was determined by flow cytometric assay after Annexin- V-PI staining. Nitric oxide levels were measured by Griess assay. Inducible nitric oxide synthase(iNOS) protein and NF-kappaBp65 fragment were detected by Western blot.</p><p><b>RESULTS</b>Exposure of 25 micromol/L t-BHP to MIN6 cells for 60 min, cell viability was reduced and the percentage of apoptosis was increased significantly. The levels of cytoplasmic iNOS protein and nitrite were elevated. Meanwhile, treatment with t-BHP resulted in nucleus NF-kappaBp65 fragment peaking at 20 min. Both L-NAME and N-Acetyl-l-cysteine (NAC) attenuated the elevated levels of nitrite and percentage of apoptosis due to t-BHP alone.</p><p><b>CONCLUSION</b>NF-kappa-iNOS-nitric oxide signalling pathway can mediated t-BHP induced apoptosis in MIN6 cells .</p>
Subject(s)
Animals , Mice , Apoptosis , Cell Line , Insulin-Secreting Cells , Cell Biology , NF-kappa B , Metabolism , Nitric Oxide , Metabolism , Nitric Oxide Synthase Type II , Metabolism , Oxidative Stress , Physiology , Signal Transduction , tert-Butylhydroperoxide , PharmacologyABSTRACT
To explore the effect of glucagon-like peptide-1 receptor agonist--Exendin-4 (Ex-4) on murine MIN6 pancreatic beta-cells apoptosis induced by oxidative stress, the morphological changes of cell damage were evaluated by epifluorescence microscopy after staining with AO-EB. The percentage of cell apoptosis was determined by flow cytometric assay after Annexin-V-FITC-PI staining. Nitric oxide level was measured by Griess reagent assay. Inducible nitric oxide synthase (iNOS) protein and NF-kappaBp65 fragment were detected by Western blotting. Ex-4 inhibited the increase of nitrite level and percentage of apoptosis induced by t-BHP in MIN6 cells. Furthermore, Ex-4 partly reduced the expression of iNOS protein and the ratio of NF-kappaBp65 protein in nucleus:cytosol induced by t-BHP. These results suggest that Ex4 protects MIN6 pancreatic kappa-cells from oxidative stress-induced apoptosis via down-regulation of NF-kappaB-iNOS-nitric oxide pathway.
Subject(s)
Animals , Mice , Apoptosis , Down-Regulation , Glucagon-Like Peptide-1 Receptor , Hypoglycemic Agents , Pharmacology , Incretins , Insulin-Secreting Cells , Cell Biology , Metabolism , Lizards , Nitric Oxide , Metabolism , Nitric Oxide Synthase Type II , Metabolism , Oxidative Stress , Peptides , Pharmacology , Receptors, Glucagon , Signal Transduction , Transcription Factor RelA , Metabolism , Venoms , Pharmacology , tert-Butylhydroperoxide , PharmacologyABSTRACT
Expression of prepro-orexin gene in the hypothalamus was decreased in rats with dietary obesity,and has no correlation with serum corticosterone concentration.Expression of orexin receptor-2 in adrenal was increased in obese rats,and was positively correlated to expression of CRH mRNA and inversely correlated to serum corticosterene concentration.