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Octanoate affects 2,4-dinitrophenol uncoupling in intact isolated rat hepatocytes
B Sibille1, C Keriel, E Fontaine
1Laboratoire de Thérapeutique, Université Joseph Fourier, Grenoble, France.
European Journal of Biochemistry
|July 15, 1995
Summary
Uncoupling rat hepatocytes with 2,4-dinitrophenol impairs reducing equivalent transport, but fatty acids restore mitochondrial function and ATP synthesis. This highlights substrate-dependent effects on cellular respiration.
Area of Science:
- Cellular Metabolism
- Mitochondrial Function
- Biochemical Energetics
Background:
- Isolated rat hepatocytes are crucial models for studying cellular metabolism.
- Mitochondrial uncoupling disrupts cellular energy production.
- Substrate availability significantly influences metabolic flux and redox state.
Purpose of the Study:
- To investigate the impact of 2,4-dinitrophenol-induced uncoupling on hepatocyte metabolism.
- To determine the role of exogenous substrates (dihydroxyacetone and octanoate) in modulating uncoupled cell responses.
- To elucidate the mechanism by which fatty acids restore mitochondrial function in uncoupled cells.
Main Methods:
- Perfusion of isolated rat hepatocytes.
- Uncoupling of mitochondria using 2,4-dinitrophenol.
- Measurement of oxygen uptake, glucose and lactate+pyruvate fluxes, cytosolic/mitochondrial redox states, and ATP/ADP ratios.
- Inhibition of the malate-aspartate shuttle with aminooxyacetate.
Main Results:
- 2,4-Dinitrophenol uncoupling with dihydroxyacetone increased cytosolic NADH/NAD+ ratio, abolished glucose production, and decreased ATP/ADP ratios.
- Octanoate addition to uncoupled cells enhanced respiration, restored glucose flux, and increased ATP/ADP ratios.
- Inhibition of the malate-aspartate shuttle reduced octanoate's effect on respiration.
Conclusions:
- 2,4-Dinitrophenol uncoupling inhibits reducing equivalent translocation into mitochondria, likely via impairing the malate-aspartate shuttle.
- Fatty acid (octanoate) addition restores mitochondrial membrane potential and protonmotive force in uncoupled cells.
- Fatty acids facilitate reducing equivalent transfer into the mitochondrial matrix, supporting respiration and ATP synthesis.