Diabetes Metab J.  2011 Oct;35(5):469-479. 10.4093/dmj.2011.35.5.469.

Protective Effect of Heme Oxygenase-1 on High Glucose-Induced Pancreatic beta-Cell Injury

• Lee EM
• Lee YE
• Lee E
• Ryu GR
• Ko SH
• Moon SD
• Song KH
• Ahn YB

• KMID: 1857502
• DOI: http://doi.org/10.4093/dmj.2011.35.5.469

Abstract

BACKGROUND
Glucose toxicity that is caused by chronic exposure to a high glucose concentration leads to islet dysfunction and induces apoptosis in pancreatic beta-cells. Heme oxygenase-1 (HO-1) has been identified as an anti-apoptotic and cytoprotective gene. The purpose of this study is to investigate whether HO-1 up-regulation when using metalloprotophyrin (cobalt protoporphyrin, CoPP) could protect pancreatic beta-cells from high glucose-induced apoptosis.
METHODS
Reverse transcription-polymerase chain reaction was performed to analyze the CoPP-induced mRNA expression of HO-1. Cell viability of INS-1 cells cultured in the presence of CoPP was examined by acridine orange/propidium iodide staining. The generation of intracellular reactive oxygen species (ROS) was measured using flow cytometry. Glucose stimulated insulin secretion (GSIS) was determined following incubation with CoPP in different glucose concentrations.
RESULTS
CoPP increased HO-1 mRNA expression in both a dose- and time-dependent manner. Overexpression of HO-1 inhibited caspase-3, and the number of dead cells in the presence of CoPP was significantly decreased when exposed to high glucose conditions (HG). CoPP also decreased the generation of intracellular ROS by 50% during 72 hours of culture with HG. However, decreased GSIS was not recovered even in the presence of CoPP.
CONCLUSION
Our data suggest that CoPP-induced HO-1 up-regulation results in protection from high glucose-induced apoptosis in INS-1 cells; however, glucose stimulated insulin secretion is not restored.

Keyword

Cobalt protoporphyrin; Diabetes mellitus; Glucotoxicity; Heme oxygenase-1

MeSH Terms

Apoptosis
Caspase 3
Cell Survival
Diabetes Mellitus
Flow Cytometry
Glucose
Heme
Heme Oxygenase-1
Insulin
Protoporphyrins
Reactive Oxygen Species
RNA, Messenger
Up-Regulation
Caspase 3
Glucose
Heme
Heme Oxygenase-1
Insulin
Protoporphyrins
RNA, Messenger
Reactive Oxygen Species

Figure

• 

  Fig. 1 mRNA expression of heme oxygenase-1 (HO-1) in INS-1 cells after 24 hours treatment with different concentration of cobalt protoporphyrin (CoPP) (0 nM, 500 pM, 5 nM, 50 nM, 500 nM, 5 µM, 50 µM). Data are mean±standard error of the 3 separate experiments. aP<0.01 vs. control (0 nM).

• 

  Fig. 2 Heme oxygenase-1 (HO-1) mRNA expression of INS-1 cells in the presence of 500 nM of cobalt protoporphyrin (CoPP) with different time course (1, 2, 4, 6, and 24 hours). Data are mean±standard error of the 3 separate experiments. aP<0.01 vs. control (0 nM).

• 

  Fig. 3 Cytotoxicity assay of qualifying cell death in INS-1 cells. 5×105 cells/well were seeded into a 96-well plate, and each well was treated with 10 µL of CCk-8 solution (Cell counting kit-8) for 3 hours at 37℃ containing 5% CO2. Data are expressed as mean±standard error of the 3 separate experiments. aP<0.05 vs. control (0 nM).

• 

  Fig. 4 Cell death rate in cultured INS-1 cells according to acridine orange (AO)/propidium iodide (PI) staining. (A) AO/PI staining in the presence of mannitol, low glucose (LG; 5.5 mM/L), and high glucose (HG; 27.7 mM/L) concentrations in INS-1 cells. The red-stained cells are dead. (B) Time-dependent course of cell death rate. Image was obtained using a microscope (×200). Data are expressed as mean±standard error of the 3 separate experiments. aP<0.05 vs. LG (72 hours).

• 

  Fig. 5 Effect of cobalt protoporphyrin (CoPP) on cell death rate in cultured INS-1 cells using acridine orange (AO)/propidium iodide (PI) staining. (A) AO/PI staining in the presence or absence of CoPP (500 nM) in INS-1 cells at different glucose concentrations. The red-stained cells represent dead cells. (B) Effect of CoPP on cell death rate in cultured INS-1 cells using AO/PI staining. Imaging performed with a microscope (×200). Data are expressed as mean±standard error of the three separate experiments. aP<0.05 vs. low glucose (LG), bP<0.01 vs. high glucose (HG).

• 

  Fig. 6 Intracellular peroxide level in INS-1 cells using flow cytometry. (A) The amount of reactive oxygen species (ROS) was measured to observe the effect of cobalt protoporphyrin (CoPP). (B) Fluorescence microscopy of ROS staining in INS-1 cells. The green staining is DCFDA and the red staining is propidium iodide. Image obtained using a microscope (×200). Data are expressed as mean±standard error of the three separate experiments. aP<0.01 vs. low glucose (LG), bP<0.05 vs. high glucose (HG).

• 

  Fig. 7 Effect of cobalt protoporphyrin (CoPP) on protein level was determined using a Western blot analysis. (A) The level of heme oxygenase-1 (HO-1) protein. (B) The level of caspase-3 protein. (C) Confocal microscopy for TUNEL staining in INS-1 cells at 72 hours. The green staining is TUNEL and the blue staining is DAPI. Imaging performed with a confocal microscope (×400). Data are expressed as mean±standard error for the 3 separate experiments. aP<0.05 vs. low glucose (LG), bP<0.01 vs. high glucose (HG).

• 

  Fig. 8 The effect of cobalt protoporphyrin (CoPP) on glucose-stimulated insulin secretion at 24 hours and 72 hours in low glucose (LG; 5.5 mM/L) and high glucose (HG; 25 mM/L) concentrations. Data are expressed as mean±standard error of the 6 separate experiments. aP<0.05 vs. LG.

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