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Objective To investigate the role of sulfydral redox agent in the modulation of insulin secretion and the potential mechanism. Methods Insulin secretion was evaluated in INS-1 cells after treatment with different concentrations of glucose and sulfydral redox agents by a standard insulin radio immunoassay. Results Glucose concentration-dependently potentiates insulin secretion was observed in INS-1 cells. DTBNP and DTDP could not only significantly increase glucose-stimulated insulin secretion (GSIS), but also increase insulin secretion in nifedipine-pretreated cells, which could be abrogated by DTT. Importantly, pharmacological ablation of L-type calcium channels by nifedipine and/or ablation of K ATP channelby diazoxide both could potentiate glucose-induced insulin secretory. Conclusions Sulfydral redox agent could regulates GSIS. DTBNP and DTDP may increase insulin secretion via regulating the activities of KATP, L-type CaV channel and IP3 receptor.
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Objective To amplify the recombinant adenovirus vector carrying rat angiotensin Ⅱ type 2 receptor (AT2R) gene using human embryonic kidney (HEK) 293A cell lines and to construct a pancreatic islet βcell model overexpressing AT2R by transfecting the adenovirus vector into rat insulinoma (INS-1) cell lines.Methods Recombinant adenovirus vector Ad-G-AT2R-EGFP and control vector Ad-CMV-EGFP were amplified with HEK 293A cells and the titer of the adenovirus was detected .After both adenovirus vectors were transfected into INS-1 cells,AT2R and angiotensin Ⅱtype 1 receptor(AT1R) gene expressions were tested using real-time PCR, Western blotting, immunofluorescence staining and confocal laser-scanning microscopy .Results The titer of amplified Ad-G-AT2R-EGFP and Ad-CMV-EGFP was re-spectively 9 ×109 pfu/ml and 8 ×109 pfu/ml.Transfection of Ad-G-AT2R-EGFP into INS-1 cells induced an increase in AT2R mRNA expression in a dose-dependent manner , and significantly increased AT2R mRNA and protein expression compared with Ad-CMV-EGFP-or mock-transfection.Conclusion The recombinant adenoviral vector carrying AT2R gene is successfully amplified and an INS-1 cell model overexpressing AT2R is constructed by transient transfection , which can contribute to further study of the role of AT2R in pancreatic islet βcells.
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BACKGROUND: Insulin receptor substrate 2 (IRS-2) is a key regulator of beta cell proliferation and apoptosis. This study was aimed to investigate effect of the glucolipotoxicity on apoptosis in INS-1 cell, and the effect of Exendin-4, a GLP-1 receptor agonist, on IRS-2 expression in the glucolipotoxicity induced INS-1 cell. The goal was to discover the new action mechanism and function of Exendin-4 in beta cell apoptosis. METHOD: INS-1 cells were cultured in glucolipotoxic condition for 2, 4 or 6 days and were categorized as G groups. Another group in which 50 nM Exendin-4 was added to INS-1 cells, cultured in glucolipotoxic condition, were named as Ex-4 groups. We investigated the expression of IRS-2 by RT-PCR, phosphorylated IRS-2 and phosphorylated Akt protein levels by western blot. We measured the apoptosis ratio of INS-1 cell in glucolipotoxic condition by TUNEL staining in both groups. RESULT: IRS-2 expression of INS-1 cells decreased with correlation to the time of exposure to glucolipotoxic condition. pIRS-2 and pAkt protein levels decreased in the similar pattern in glucolipotoxicity group. However, this effect of glucolipotoxicity on INS-1 cell was inhibited by the Exendin-4 treatment. In the Ex-4 groups, IRS-2 expression, pIRS-2 and pAkt protein levels remained at the similar level to low glucose condition state. Also, apoptosis induced by glucolipotoxicity was suppressed by Exendin-4 treatment significantly. CONCLUSION: We showed that the long-term treatment of Exendin-4 inhibited the apoptosis of beta cells significantly in glucolipotoxic condition and that this effect of Exendin-4 was related with IRS-2 and Akt among the beta cell's intracellular signal transduction pathway.
Subject(s)
Apoptosis , Blotting, Western , Cell Proliferation , Cells, Cultured , Glucagon-Like Peptide 1 , Glucagon-Like Peptide-1 Receptor , Glucose , In Situ Nick-End Labeling , Insulin Receptor Substrate Proteins , Peptides , Phosphorylation , Receptors, Glucagon , Signal Transduction , VenomsABSTRACT
BACKGROUND: The highly developed endoplasmic reticulum (ER) structure is one of the characteristic features of pancreatic beta-cells. Recent study showed that ER stress causes beta-cell dysfunction. However, little is known about the effects of high glucose concentration on induction of ER stress in pancreatic beta-cells. Therefore, this study was designed to evaluate whether exposure of high glucose concentration in rat insulinoma cell line, INS-1 cell induces ER stress and whether ER stress decreases insulin gene expression. METHODS: The effect of 30 mM glucose on insulin expression and secretion in INS-1 cells was evaluated by Northern blot analysis and glucose-stimulated insulin secretion (GSIS). Cell viability was evaluated by XTT assay. The effect of 30 mM glucose on phosphorylation of eIF2alpha and CHOP expression, which are markers of ER stress were evaluated by Western blot analysis. RT-PCR analysis was performed to determine whether high glucose concentration induces XBP-1 splicing. To investigate whether ER stress decreases insulin gene expression, the effect of tunicamycin on insulin mRNA expression was evaluated by Northern blot analysis. RESULTS: The prolonged exposure of INS-1 cells with the 30 mM glucose concentration decreased insulin mRNA expression in a time dependent manner and impaired GSIS while did not influence on cell viability. 30 mM glucose increased phosphorylation of eIF2alpha, XBP-1 splicing and CHOP expression in INS-1 cells. Tunicamycin-treated INS-1 increased XBP-1 splicing and decreased insulin mRNA expression in a dose dependent manner. CONCLUSION: This study showed that prolonged exposure of INS-1 with high glucose concentration induces ER stress and ER stress decreases insulin gene expression. Further studies about underlying molecular mechanism by which ER stress induces beta-cell dysfunction are needed.
Subject(s)
Animals , Rats , Blotting, Northern , Blotting, Western , Cell Line , Cell Survival , Endoplasmic Reticulum , Endoplasmic Reticulum Stress , Gene Expression , Glucose , Hyperglycemia , Insulin , Insulinoma , Phosphorylation , RNA, Messenger , TunicamycinABSTRACT
0.05),Glibenclamide(0.1?mol/L,1?mol/L,10?mol/L) increased cell′s apoptosis by2.35,2.71,2.94 folders(P
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Aim To investigate the protective effects of polysaccharides from the rhizomorph of Armillaria mella(AMP-1) on alloxan injured INS-1 cells.Methods Graded concentrations of AMP-1(2,10,50,100,500,1 000 mg?L-1) were added into the culture medium of alloxan injured INS-1 cells.The survival rate was measured by MTT assay.The amount of glucose-stimulated insulin secretion in different concentrations of AMP-1 was determined by radioimmunoassay(RIA).SOD and NOS activity,NO,MDA and GSH production were assayed colorimetrically.Results AMP-1 could reduce oxidative injuries induced by alloxan in INS-1 cells.The survival rate of cells treated with AMP-1 increased significantly.In the presence of 5.6 mmol?L-1 or 16.7 mmol?L-1 glucose,AMP-1(50,100,500,1 000 mg?L-1)increased glucose-induced insulin secretion in INS-1 cells in a dose-dependent manner.NOS levels and the production of NO and MDA decreased significantly by AMP-1,while SOD levels and the production of GSH increased.Conclusions AMP-1 promoted glucose-induced insulin secretion in INS-1 cells by increasing the abilities of scavenging the free radicals induced by alloxan.
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In INS-1 cells, the insulin secretion was investigated by radioimmunoassay (RIA) after 4 h incubation in medium containing different concentrations of glucose and recombinant human glucagon-like peptide-1 (rhGLP-1) (7-36). Insulin mRNA level in INS-1 cells was assessed by a semi-quantitative RT-PCR method. rhGLP-1 (7-36) is not only a powerful insulin secretagogue, but also can increase insulin gene expression in INS-1 cells.