Korean Diabetes J.  2008 Apr;32(2):102-111. 10.4093/kdj.2008.32.2.102.

The Effects of Exendin-4 on IRS-2 Expression and Phosphorylation in INS-1 Cells

• Kim JH
• Kim JW
• Jeon SY
• Park HS
• Ham DS
• You YH
• Lee SH
• Cho JH
• Kang MJ
• Lee KW
• Kwon HS
• Yoon KH
• Cha BY
• Lee KW
• Kang SK
• Son HY

• KMID: 2222506
• DOI: http://doi.org/10.4093/kdj.2008.32.2.102

Abstract

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.

Keyword

Exenatide; Insulin receptor substrate 2; INS-1 cell

MeSH Terms

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
Venoms
Glucagon-Like Peptide 1
Glucose
Insulin Receptor Substrate Proteins
Peptides
Receptors, Glucagon
Venoms

Figure

• 

  Fig. 1 Schematic of experimental design. A. Apoptosis measurement of isolated islet cell and INS-1 cell in glucolipotoxicity condition by AO/PI and TUNEL staining. B. Insulin gene expression of isolated islet cell in glucolipotoxicity condition. C. Confirmation of Exendin-4 concentration for INS-1 cell treatment using cAMP assay in three concentration group (Ex-4 0, 10, 100 nM). D. General scheme of INS-1 cell culture were seperated into four groups: low glucose condition, low glucose + 50 nM Exendin-4, Glucolipotoxic condition (GLTx.), GLTx + 50 nM Exendin-4. Insulin and IRS-2 gene expression was measured by RT-PCR and phosphorylated IRS-2 and Akt protein was measured by western blot. E. Apoptosis of INS-1 cell in GLTx group and GLTx + 50 nM Exendin-4 group were measured by TUNEL staining.

• 

  Fig. 2 Apoptosis of the isolated islet cell in glucolipotoxic condition. A. Apoptosis according to the exposure time (2, 4, 8 days) in AO/PI. B. Apoptosis of INS-1 cell in TUNEL staining (arrow: apoptotic cells). Few islet cells were survived in glucolipotoxic condition during 8 days in glucolipotoxic condition (×400).

• 

  Fig. 3 Effect of the glucolipotoxic condition on insulin gene expression in isolated islet cells. Insulin was estimated by RT-PCR. GLTx: glucolipotoxicity.

• 

  Fig. 4 Effect of exendin-4 concentration on Cyclic AMP levels in INS-1 cells. There was little difference in mean value of cAMP levels between 0 nM and 10 nM of Exendin-4 concentration. But, the mean value of cAMP levels in 100 nM of Exendin-4 was significantly higher than that of 10 nM of Exendin-4 concentration. *P < 0.05 vs. Ex-4 0 nM.

• 

  Fig. 5 IRS-2 and insulin gene expression on glucolipotoxic condition vs 50 nM exendin-4 treatment group in INS-1 cells. IRS-2 and insulin gene expression were increased in Exendin-4 treatment group compared with control and glucolipotoxic condition.

• 

  Fig. 6 Phosphorylated IRS-2 and pAkt protein levels in glucolipotoxic condition and effect of the exendin-4 on pIRS-2 and pAkt. A. pIRS-2 and pAkt were increased in Exendin-4 treatment group according to the exposure duration compared with the control group. B. Protective effect of the Exendin-4 treatment that inhibited pIRS-2 and pAkt protein reduction in glucolipotoxic condition.

• 

  Fig. 7 The effect of Exendin-4 on apoptosis in glucolipotoxic condition. A INS-1 cell apoptosis was progressed in glucolipotoxic condition according to prolongation of exposure but, Exendin-4 treatment in the same condition inhibited apoptosis in TUNEL staining (×400). B. The apptosis rate was decreased in Exendin-4 treatment condition significantly (*P < 0.05, †P < 0.01).

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