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Mitochondrial DNA in beta-cells is a sensitive target for damage by nitric oxide
G L Wilson1, N J Patton, S P LeDoux
1Department of Structural and Cellular Biology, The University of South Alabama, Mobile 36688, USA.
Abstract:
Increasing evidence indicates that nitric oxide (NO) may play a role in immune-mediated injury to beta-cells. One site for the action of this agent is the mitochondrion. Although the exact targets for damage within this organelle have yet to be fully elucidated, a potential location for injury is mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by NO. Both exogenous NO generation (spermine/NO adduct [sper/NO]) and endogenous production of NO (IL-1beta) were studied. To study the effects of exogenously produced NO, neonatal rat islet cells in monolayers were exposed to varying doses of sper/NO for 30 min. Total cellular DNA was isolated and treated with alkali to produce strand breaks at abasic sites resulting from exposure to NO. Damage to mtDNA was evaluated using a quantitative Southern blot technique. The results showed that sper/NO caused dose-dependent damage to mtDNA. Additionally, mtDNA was found to be more sensitive to injury generated by either source than a similarly sized fragment of nuclear DNA. To evaluate the effects of endogenously produced NO, beta-cell cultures were treated with IL-1beta for 18 h. Other cultures were treated with IL-1beta and an inhibitor of the inducible form of nitric oxide synthase, aminoguanidine. DNA was evaluated as described for the sper/NO studies. IL-1beta caused appreciable damage to mtDNA, and this damage was reduced in mtDNA from cultures treated with IL-1beta and aminoguanidine. These studies show that mtDNA is a sensitive target for NO generated both endogenously and exogenously and that this DNA is more vulnerable to NO-induced damage than nuclear DNA.
Insights
Nitric oxide (NO) damages mitochondrial DNA (mtDNA) in beta-cells. This study shows mtDNA is a sensitive target for NO, more vulnerable than nuclear DNA, impacting immune-mediated injury.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Nitric oxide (NO) is implicated in immune-mediated beta-cell injury.
- Mitochondria are a key site of NO action within beta-cells.
- Mitochondrial DNA (mtDNA) is a potential target for NO-induced damage.
Purpose of the Study:
- To investigate the damaging effects of nitric oxide on mitochondrial DNA (mtDNA).
- To compare the susceptibility of mtDNA versus nuclear DNA to NO-induced damage.
- To evaluate NO-induced mtDNA damage from both exogenous and endogenous sources.
Main Methods:
- Neonatal rat islet cells were exposed to exogenous spermine/NO (sper/NO).
- Beta-cell cultures were treated with interleukin-1beta (IL-1beta) to induce endogenous NO production.
- DNA damage was quantified using alkali treatment and Southern blot analysis.
- Inhibitor of inducible nitric oxide synthase (aminoguanidine) was used to assess endogenous NO effects.
Main Results:
- Exogenous sper/NO caused dose-dependent damage to mtDNA.
- mtDNA exhibited greater sensitivity to NO-induced damage compared to nuclear DNA.
- Endogenous NO production via IL-1beta induced significant mtDNA damage.
- Aminoguanidine treatment reduced IL-1beta-induced mtDNA damage.
Conclusions:
- Mitochondrial DNA (mtDNA) is a sensitive target for nitric oxide (NO).
- NO generated both exogenously and endogenously can damage mtDNA.
- mtDNA is more vulnerable to NO-induced injury than nuclear DNA, suggesting a role in beta-cell dysfunction.