Korean Diabetes J.  2010 Oct;34(5):312-319. 10.4093/kdj.2010.34.5.312.

The Effect of Tribbles-Related Protein 3 on ER Stress-Suppressed Insulin Gene Expression in INS-1 Cells

• Jang YY
• Kim NK
• Kim MK
• Lee HY
• Kim SJ
• Kim HS
• Seo HY
• Lee IK
• Park KG

• KMID: 2029889
• DOI: http://doi.org/10.4093/kdj.2010.34.5.312

Abstract

BACKGROUND
The highly developed endoplasmic reticulum (ER) structure in pancreatic beta cells is heavily involved in insulin biosynthesis. Thus, any perturbation in ER function inevitably impacts insulin biosynthesis. Recent studies showed that the expression of tribbles-related protein 3 (TRB3), a mammalian homolog of Drosophilia tribbles, in various cell types is induced by ER stress. Here, we examined whether ER stress induces TRB3 expression in INS-1 cells and found that TRB3 mediates ER stress-induced suppression of insulin gene expression.
METHODS
The effects of tunicamycin and thapsigargin on insulin and TRB3 expression in INS-1 cells were measured by Northern and Western blot analysis, respectively. The effects of adenovirus-mediated overexpression of TRB3 on insulin, PDX-1 and MafA gene expression in INS-1 cells were measured by Northern blot analysis. The effect of TRB3 on insulin promoter was measured by transient transfection study with constructs of human insulin promoter.
RESULTS
The treatment of INS-1 cells with tunicamycin and thapsigargin decreased insulin mRNA expression, but increased TRB3 protein expression. Adenovirus-mediated overexpression of TRB3 decreased insulin gene expression in a dose-dependent manner. A transient transfection study showed that TRB3 inhibited insulin promoter activity, suggesting that TRB3 inhibited insulin gene expression at transcriptional level. Adenovirus-mediated overexpression of TRB3 also decreased PDX-1 mRNA expression, but did not influence MafA mRNA expression.
CONCLUSIONS
This study showed that ER stress induced TRB3 expression, but decreased both insulin and PDX-1 gene expression in INS-1 cells. Our data suggest that TRB3 plays an important role in ER stress-induced beta cell dysfunction.

Keyword

Diabetes; Endoplasmic reticulum stress; Insulin; TRB3

MeSH Terms

Blotting, Northern
Blotting, Western
Endoplasmic Reticulum
Endoplasmic Reticulum Stress
Gene Expression
Humans
Insulin
Insulin-Secreting Cells
RNA, Messenger
Thapsigargin
Transfection
Tunicamycin
Insulin
RNA, Messenger
Thapsigargin
Tunicamycin

Figure

• 

  Fig. 1 The effects of tunicamycin and thapsigargin on insulin mRNA expression in INS-1 cells. Northern blot analysis of insulin mRNA expression in INS-1 cells treated with tunicamycin (A) and thapsigargin (B). INS-1 cells were treated with tunicamycin (2 µg/mL) for 24 hours or thapsigargin (1 µM) for 5 hours. 18S rRNA levels were analyzed as an internal control. Data in bar graph are the mean ± SEM of three independent measurements. aP < 0.01 and bP < 0.001 compared to control.

• 

  Fig. 2 The effects of tunicamycin and thapsigargin on TRB3 protein expression in INS-1 cells. Western blot analysis of TRB3 protein expression in the presence of tunicamycin (A) and thapsigargin (B). INS-1 cells were incubated with tunicamycin (2 µg/mL) and thapsigargin (1 µM) for indicated times. β-actin protein levels were analyzed as an internal control. The data in bar graph are the mean ± SEM of three independent measurements. aP < 0.01 and bP < 0.001 compared to 0 hour.

• 

  Fig. 3 The effects of adenovirus-mediated overexpression of TRB3 on insulin, PDX-1 and MafA mRNA expression. Northern blot analysis of insulin, PDX-1 and MafA mRNA expression in INS-1 cells infected with adenovirus encoding TRB3 (Ad-TRB3). INS-1 cells were infected with the indicated doses of Ad-TRB3 or Ad-LacZ for 24 hours. 18S rRNA levels were analyzed as an internal control. Data in the bar graph are the mean ± SEM of three independent measurements. aP < 0.01 and bP < 0.001 compared to control.

• 

  Fig. 4 The effects of TRB3 on insulin promoter activity. INS-1 cells were cotransfected with the human insulin promoter (200 ng/well) and the indicated amounts of an expression vector for TRB3 for 24 hours. Data represent the mean ± SEM of three independent measurements. aP < 0.01 and bP < 0.001 compared to control.

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