J Korean Diabetes Assoc.  2007 Mar;31(2):105-112. 10.4093/jkda.2007.31.2.105.

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

• Koh G
• Woo JT
• Lee DH
• Oh S
• Kim SW
• Kim JW
• Kim YS
• Park DB

• KMID: 2008078
• DOI: http://doi.org/10.4093/jkda.2007.31.2.105

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 beta-cell line. We performed this study to investigate the mechanism of dRib-induced damage of beta-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 beta-cell. Thus, dRib could be a surrogate for glucose in the study of glucose toxicity and chronic diabetic complications.

Keyword

Autoxidation; Glucose toxicity; Oxidative stress; Protein glycation; beta-cell apoptosis; 2-deoxy-D-ribose

MeSH Terms

Apoptosis
Cell Survival
Diabetes Complications
Flow Cytometry
Glucose
Oxidative Stress
Pentetic Acid
Pyridoxamine
Glucose
Pentetic Acid
Pyridoxamine

Figure

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  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.

• 

  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.

• 

  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.

• 

  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.

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