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ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition
A L Nieminen1, A K Saylor, B Herman
1Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill 27599-7090.
Abstract:
The importance of ATP depletion and mitochondrial depolarization in the toxicity of cyanide, oligomycin, and carbonyl cyanide m-cholorophenylhydrazone (CCCP), an uncoupler, was evaluated in rat hepatocytes. Oligomycin, an inhibitor of the reversible mitochondrial ATP synthase (F1F0-adenosinetriphosphatase), caused dose-dependent cell killing with 0.1 microgram/ml being the minimum concentration causing the maximum cell killing. Oligomycin also caused rapid ATP depletion without causing mitochondrial depolarization. Fructose (20 mM), a potent glycolytic substrate in liver, protected completely against oligomycin toxicity. CCCP (5 microM) also caused rapid killing of hepatocytes. Fructose retarded cell death caused by CCCP but failed to prevent lethal cell injury. Although oligomycin (1.0 microgram/ml) was lethally toxic by itself, in the presence of fructose it protected completely against CCCP-induced cell killing. Cyanide (2.5 mM), an inhibitor of mitochondrial respiration, caused rapid cell killing that was reversed by fructose. CCCP completely blocked fructose protection against cyanide, causing mitochondrial depolarization and rapid ATP depletion. In the presence of fructose and cyanide, oligomycin protected cells against CCCP-induced ATP depletion and cell death but did not prevent mitochondrial depolarization. In every instance, cell killing was associated with ATP depletion, whereas protection against lethal cell injury was associated with preservation of ATP. In conclusion, protection by fructose against toxicity of cyanide, oligomycin, and CCCP was mediated by glycolytic ATP formation rather than by preservation of the mitochondrial membrane potential. These findings support the hypothesis that inhibition of cellular ATP formation is a crucial event in the progression of irreversible cell injury.
Insights
Fructose protects rat hepatocytes from toxic agents by boosting ATP production through glycolysis, not by preserving mitochondrial function. This highlights ATP depletion as key to cell injury.
Area of Science:
- Hepatocyte toxicology
- Mitochondrial function
- Cellular energy metabolism
Background:
- Cyanide, oligomycin, and CCCP are known to induce toxicity through cellular energy disruption.
- The roles of ATP depletion and mitochondrial depolarization in this toxicity require further elucidation.
- Fructose is a potent glycolytic substrate in liver cells.
Purpose of the Study:
- To evaluate the importance of ATP depletion and mitochondrial depolarization in the toxicity of cyanide, oligomycin, and CCCP in rat hepatocytes.
- To investigate the protective mechanisms of fructose against these toxic agents.
Main Methods:
- Toxicity assays were performed on rat hepatocytes exposed to cyanide, oligomycin, and CCCP.
- ATP levels and mitochondrial membrane potential were measured.
- The effects of fructose, a glycolytic substrate, were assessed in the presence of these toxins.
Main Results:
- Oligomycin caused dose-dependent cell killing and ATP depletion without mitochondrial depolarization; fructose provided complete protection.
- CCCP induced rapid hepatocyte killing, which was only partially retarded by fructose.
- Fructose protected against cyanide toxicity, but this protection was abolished by CCCP, which also caused mitochondrial depolarization and ATP depletion.
Conclusions:
- Cell killing was consistently associated with ATP depletion, while protection correlated with ATP preservation.
- Fructose-mediated protection against cyanide, oligomycin, and CCCP toxicity is primarily due to enhanced glycolytic ATP formation, not mitochondrial membrane potential preservation.
- Inhibition of cellular ATP formation is a critical factor in the progression of irreversible cell injury.