Korean Diabetes J.  2008 Apr;32(2):121-130. 10.4093/kdj.2008.32.2.121.

The Protective Effect of EGCG on INS-1 Cell in the Oxidative Stress and Mechanism

• Kim MK
• Jung HS
• Yoon CS
• Kwon MJ
• Koh KS
• Rhee BD
• Park JH

• KMID: 2222508
• DOI: http://doi.org/10.4093/kdj.2008.32.2.121

Abstract

BACKGROUND: Oxidative stress is important in both diabetic complications and the development and the progression of type 2 diabetes via the effects on the pancreatic beta-cells. EGCG (epigallocatechin galleate), a major constituent of green tea, has been known to have beneficial effects on various diseases through the mechanisms of antioxidant and cell signaling modulation. But, very small numbers of studies were published about the direct effects of EGCG on the pancreatic beta cell lines. We performed this study to see the protective effect of EGCG on pancreatic beta cell line under H2O2 and the mechanisms of this phenomenon.
METHODS
We used INS-1 cells and hydrogen peroxide as an oxidative stressor. Their viabilities were verified by MTT assay and FACS. The activity of glutathione peroxidase was assessed by total glutathione quantification kit. Western blot and semi-quantitative RT-PCR for the catalase, SOD (superoxide dismutase), PI3K and Akt were performed. Functional status of INS-1 cells was tested by GSIS (glucose stimulated insulin secretion).
RESULTS
The biological effects of EGCG were different according to its concentrations. 10 micrometer EGCG effectively protected hydrogen peroxide induced damage in INS-1 cells. The expression and the activity of SOD, catalase and the glutathione peroxidase were significantly increased by EGCG. EGCG significantly increased PI3K and Akt activity and its effect was inhibited partially by wortmannin. GSIS was well preserved by EGCG.
CONCLUSION
EGCG in low concentration effectively protected INS-1 cells from the oxidative stress through the activation of both antioxidant systems and anti-apoptosis signaling. Further studies will be necessary for the more detailed mechanisms and the clinical implications.

Keyword

Antiapoptosis; Antioxidant; EGCG; INS cell; Oxidative stress

MeSH Terms

Androstadienes
Blotting, Western
Catalase
Diabetes Complications
Glutathione
Glutathione Peroxidase
Hydrogen Peroxide
Insulin
Insulin-Secreting Cells
Oxidative Stress
Tea
Androstadienes
Catalase
Glutathione
Glutathione Peroxidase
Hydrogen Peroxide
Insulin
Tea

Figure

• 

  Fig. 1 The cell viability assessed by MTT assay (A) and FACS (B). Cell viability against H2O2 (80 µM) induced toxicity in INS-1 cells was different on the concentration of EGCG. In the histogram, the results obtained from four independent experiments are reported as means ± S.D. (A). FACS analysis after double staining with annexin V/propidium iodide (FL1:AnnexinV, FL2:PI). Dot plots from a representative FACS experiment are shown (B).

• 

  Fig. 2 EGCG changes antioxidant enzymes expression. Total RNA was isolated from INS-1 cells incubated for 24 h without (control; C) or with hydrogen peroxide (H2O2) and in the presence of EGCG (E). (A) Expression of MnSOD, catalase, and endogenous control GAPDH was evaluated by RT-PCR. A representative experiment of three is shown. (B) MnSOD and Catalase expression evaluated by western blot analysis. A representative experiment of three is shown. (C) Densitometric analyses of western blot are reported as means ± S.D. of the three different experiments. (D) GPx activity was evaluated by Glutathione peroxidase assay kit.

• 

  Fig. 3 EGCG modulated cell signalings related to the apoptosis. Total RNA was isolated from INS-1 cells incubated for 24 h without (control; C) or with hydrogen peroxide (H2O2) and in the presence of EGCG (E). (A) Expression of PI3K, Akt, total caspase 3, and endogenous control beta-actin was evaluated by RT-PCR. A representative experiment of three is shown. (B) Phosphorylation of PI3K and Akt and total caspase 3 were evaluated by western blot analysis. A representative experiment of three is shown. (C) Densitometric analyses of western blot are reported as means ± S.D. of the three different experiments

• 

  Fig. 4 The cell viability assessed by FACS after treatment of PI3K inhibitor. Cell viability against H2O2 (80 µM) induced toxicity in INS-1 cells was increased with 10 µM of EGCG (E) and partially decreased with PI3K inhibiotor (E+W). FACS analysis after double staining with annexin V/propidium iodide(FL1:AnnexinV, FL2:PI). Dot plots from a representative FACS experiment are shown (A). In the histogram, the results obtained from three independent experiments are reported as means ± S.D. (B).

• 

  Fig. 5 Insulin secretion from INS-1cells in response to glucose (5 and 25 mM) concentration after a 24-h incubation with control medium (control), medium containing H2O2 with EGCG (E) and without EGCG. Data are means ± S.D. of three separate experiments.

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