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Koh, Woo, Lee, Oh, Kim, Kim, Kim, and Park: Mechanism of 2-Deoxy-D-ribose-induced Damage in Pancreatic β-cells
Original Article
The Journal of Korean Diabetes Association

The Journal of Korean Diabetes Association 2007; 31(2): 105-112.

Published online: 31 March 2007

DOI: https://doi.org/10.4093/jkda.2007.31.2.105

Mechanism of 2-Deoxy-D-ribose-induced Damage in Pancreatic β-cells

Gwanpyo Koh, Jeong-taek Woo2, Dae-Ho Lee, Seungjoon Oh2, Sung-Woon Kim2, Jin-Woo Kim2, Young-Seol Kim2, Deok-Bae Park1

Department of Internal Medicine, Cheju National University College of Medicine, Korea.

1Department of Medicine, Cheju National University College of Medicine, Korea.

2Department of Endocrinology & Metabolism, Kyung Hee University College of Medicine, Korea.

Received 26 October 2006       Accepted 15 January 2007

Copyright © 2007 Korean Diabetes Association

Abstract

Background

Mechanism for glucose toxicity is known to be an increased oxidative stress produced by multiple pathways. In our previous report, 2-deoxy-d-ribose (dRib) promoted apoptosis by increasing oxidative stress in a pancreatic β-cell line. We performed this study to investigate the mechanism of dRib-induced damage of β-cells.

Methods

HIT-T15 cells were cultured in RPMI-1640 medium with 40 mM dRib for 24 hours after pretreatment with various concentrations of a metal chelator (DTPA) and inhibitors of protein glycation (aminoguanidine and pyridoxamine). Cell viability was determined by MTT assay. Apoptosis was analyzed by flow cytometry with annexin V/PI double staining.

Results

DTPA, which inhibits the monosaccharide autoxidation, partially reversed dRib-induced cytotoxicity in a dose-dependent manner (P < 0.01). The cytotoxicity was also suppressed dose-dependently by aminoguanidine (AG) and pyridoxamine (PM) (P < 0.05 and P < 0.01, repectively). Flow cytometric analysis showed that pretreatment of DTPA and AG also reversed the dRib-triggered apoptosis in a dose-dependent manner. We assessed the additional protective effects of inhibitors of protein glycation from dRib-induced cytotoxiciy in the presence of a metal chelator. The additions of AG (P < 0.05) and PM (P < 0.01) significantly reduced the cytotoxicity compared with DTPA alone group.

Conclusion

This results suggest that dRib produce cytotoxicity and apoptosis through the mechanisms of advanced glycation endproducts (AGEs) formation including the monsaccharide autoxidation and protein glycation in pancreatic β-cell. Thus, dRib could be a surrogate for glucose in the study of glucose toxicity and chronic diabetic complications.

Keywords: Autoxidation, Glucose toxicity, Oxidative stress, Protein glycation, β-cell apoptosis, 2-deoxy-D-ribose

Figures and Tables

Fig. 1
Protective effects of DTPA, aminoguanidine (AG) and pyridoxamine (PM) on dRib-triggered cytotoxicity. HIT-T15 cells were preincubated with DTPA, AG and PM for 30 min at the indicated concentrations and then cultured with 40 mM dRib for 24 h. Cell viability was determined by MTT assay. Data are expressed as the mean ± SD of the percentage of viable cells relative to the untreated control. This experiment was performed twice, in quadruplicate.
**P < 0.01 vs. control.
†P < 0.05 vs. 40 mM dRib alone.
††P < 0.01 vs. 40 mM dRib alone.
jkda-31-105-g001
Fig. 2
Effect of DTPA on dRib-induced apoptosis of HIT-T15 cells. Cells were preincubated with 0.01, 0.1 and 0.3 mM DTPA for 30 min, then cultured with 40 mM dRib for 24 h. Cells were stained with Annexin V-FITC (horizontal axis)/PI (longitudinal axis) and analyzed by flow cytometry. The graph is representative of two independent experiments. (A) Control; (B) 40 mM dRib; (C) 40 mM dRib + 0.01 mM DTPA; (D) 40 mM dRib + 0.1 mM DTPA; (E) 40 mM dRib + 0.3 mM DTPA.
jkda-31-105-g002
Fig. 3
Effect of AG on dRib-induced apoptosis of HIT-T15 cells. Cells were preincubated with 0.5, 1 and 5 mM AGfor 30 min, then cultured with 40 mM dRib for 24 h. Cells were stained with Annexin V-FITC (horizontal axis)/PI (longitudinal axis) and analyzed by flow cytometry. The graph is representative of two independent experiments. (A) Control; (B) 40 mM dRib; (C) 40 mM dRib + 0.5 mM AG; (D) 40 mM dRib + 1 mM AG; (E) 40 mM dRib + 5 mM AG.
jkda-31-105-g003
Fig. 4
Additive effects of AG and PM on DTPA-mediated protection from of dRib-induced cytotoxicity. HIT-T15 cells were preincubated with DTPA, AG and PM for 30 min at the indicated concentrations and then cultured with 40 mM dRib for 24 h. Cell viability was determined by MTT assay. Data are expressed as the mean ± SD of the percentage of viable cells relative to the untreated control. This experiment was performed twice, in quadruplicate.
**P < 0.01 vs. control.
††P < 0.01 vs. 40 mM dRib alone.
‡P < 0.05 vs. 40 mM dRib + 0.3 mM DTPA.
‡‡P < 0.01 vs. 40 mM dRib + 0.3 mM DTPA.
jkda-31-105-g004

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