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Abstract
Background
The highly developed endoplasmic reticulum (ER) structure is one of the characteristic features of pancreatic β-cells. Recent study showed that ER stress causes β-cell dysfunction. However, little is known about the effects of high glucose concentration on induction of ER stress in pancreatic β-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 eIF2α 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 eIF2α, 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.
Figures and Tables
Fig. 1
Effect of high glucose concentration on insulin mRNA expression. A. Northern blot analysis of insulin expression in the presence of high glucose. INS-1 cells were incubated with 30 mM glucose for increasing lengths of time, and mRNAs of the insulin gene were examined. 18S rRNA levels were analyzed as an internal control. B. Data represents the means ± SE of three independent measurements. *P < 0.01 and **P < 0.001 compared to 0 h. C. Effect of chronic exposure to high glucose on glucose-stimulated insulin secretion (GSIS). INS-1 cells were incubated with 5 mM and 30 mM glucose for 96 h, and then GSIS was measured as described in methods. *P < 0.01 compared to 5 mM glucose. D. INS-1 cells were treated with 30 mM glucose for increasing lengths of time and cell viability was determined by XTT assay.
Fig. 2
Effect of high glucose concentration on phosphorylation of eIF2α. A. Western blot analysis of phosphorylated eIF2α in the presence of high glucose. INS-1 cells were incubated with 30 mM glucose for the indicated times. β-actin protein levels were analyzed as an internal control. B. Data represents the means ± SE of three independent measurements. *P < 0.01 and *#x002A;P < 0.001 compared to 0 h.
Fig. 3
Effect of high glucose concentration on XBP-1 splicing. A. RT-PCR analysis of XBP-1 splicing in the presence of high glucose. INS-1 cells were incubated with 30 mM glucose for the indicated time and mRNA levels at each time point were assessed. β-actin mRNA levels were analyzed as an internal control. B. Data represents the means ± SE of three independent measurements. *P < 0.01 and **P < 0.001 compared to 0 h.
Fig. 4
Effect of high glucose concentration on CHOP expression. A. Western blot analysis of CHOP in the presence of high glucose. INS-1 cells were incubated with 30 mM glucose for the indicated times. B. Data represents the means ± SE of three independent measurements. *P < 0.01 and **P < 0.001 compared to 0 h.
Fig. 5
Effect of tunicamycin on insulin mRNA expression. A. RT-PCR analysis of XBP-1 splicing in the presence of tunicamycin. INS-1 cells were incubated with 10 ug/mL of tunicamycin for 24 h. β-actin mRNA levels were analyzed as an internal control. B. Northern blot analysis of insulin mRNA expression in cells treated with tunicamycin. INS-1 cells were incubated with indicated concentrations of tunicamycin for 24 h. 18S rRNA levels were analyzed as an internal control. Data represents the means ± SE of three independent measurements (lower panel). *P < 0.001 compared to untreated cells.
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