Octanoate Affects 2,4‐Dinitrophenol Uncoupling in Intact Isolated Rat Hepatocytes

Abstract

When intact isolated rat hepatocytes, either incubated or perifused, were uncoupled by 2,4‐dinitrophenol, we found that the effect on glucose and lactate+pyruvate fluxes, cytosolic and mitochondrial redox states and ATP/ADP ratios were dependent on the nature of the exogenous substrate added. 2,4‐Dinitro‐phenol addition (0.25 mmol/1) to cells perifused with dihydroxyacetone (10 mmol/1) resulted in a modest and transient activation of oxygen uptake accompanied by a surprising rise in lactate/pyruvate ratio indicating an increase in the cytosolic NADH/NAD+ ratio. In addition, such uncoupling, fully abolished glucose production, enhanced lactate+pyruvate flux, and strongly decreased cytosolic and mitochondrial ATP/ADP ratios. In these steady‐state conditions, further addition of Octanoate (0.4 mmol/1) induced a large and sustained enhancement of respiration with a concomitant decrease in the lactate/pyruvate ratio, whereas glucose flux was restored to some extent and cytosolic and mitochondrial ATP/ADP ratios increased. Inhibition of the malate‐aspartate shuttle by the transaminase inhibitor aminooxyacetate (0.3 mmol/1) did not modify the effect of 2,4‐dinitrophenol with dihydroxyacetone alone whereas it decreased the maximal stimulation of oxygen uptake after octanoate addition. In view of these results we propose the following conclusions. The uncoupling of intact cells by 2,4‐dinitrophenol inhibits the translocation of reducing equivalents into the mitochondrial matrix probably by impairing the malate‐aspartate shuttle. This explains the increase in the cytosolic NADH/NAD4 ratio and the transient activation of respiration with dihydroxyacetone. Fatty acid addition to cells uncoupled with 2,4‐dinitrophenol appears to restore a mitochondrial membrane potential, probably by providing the respiratory chain with reduced cofactors directly in the matrix, thus allowing the transfer of reducing equivalents across the mitochondrial membrane. The restoration, to some extent, of a protonmotive force to uncoupled cells by fatty acid addition is also supported by an increase in ATP synthesis as evidenced by a glucose synthesis with dihydroxyacetone as gluconeogenic substrate.

The effects of 2,4‐dinitrophenol and d‐glucose concentration on the development, sex ratio, and interferon‐tau (IFNT) production of bovine blastocysts

“…DNP, as an uncoupler of OXPHOS, decreases ROS production via the electron transport chain (Okuda et al, 1992;Korshunov et al, 1997) and modulates nicotinamide adenine dinucleotide (phosphate) ratios (NAD(P) H-to-NAD(P) ratio) (Sibille et al, 1995;Leverve et al, 1998;Rex et al, 1999;Hoffmann et al, 2001), thereby altering the cellular redox state to increase ATP production by glycolysis (O'Fallon and Wright 1986;Hewitson et al, 1996). Conflicting reports exist as to whether or not DNP benefits development, though.…”

Alteration of mitochondrial efficiency affects oxidative balance, development and growth in frog (Rana temporaria) tadpoles

“…Another aspect of 2,4-dNP-induced mild mitochondrial uncoupling is the increased cost of mitochondrial ATP production that could ultimately decrease the cellular availability of ATP (Sibille et al, 1995;Geelen et al, 2008). In this context, there would be three main cellular ways to compensate for the reduced mitochondrial ATP resulting from 2,4-dNP treatment: activation of glycolysis, mitochondrial biogenesis and/or increase of the oxidative phosphorylation capacity of individual mitochondria.…”

“…Chronic 2,4-dNP exposure at a low dose is expected to induce mild mitochondrial uncoupling (Wallace and Starkov, 2000). Consequently, the treatment could have a beneficial effect on oxidative stress and decrease the cellular availability of ATP (Sibille et al, 1995;Wallace and Starkov, 2000;da Silva et al, 2008;Geelen et al, 2008). There are three main ways to compensate for the reduced mitochondrial ATP production: (i) activation of the glycolytic pathway, (ii) increased mitochondrial biogenesis, and (iii) increased oxidative phosphorylation capacity of individual mitochondria.…”

AOP Report: Uncoupling of Oxidative Phosphorylation Leading to Growth Inhibition via Decreased Cell Proliferation

“…Consequently, specifically designed (eco)toxicological studies with known uncouplers as the stressors, such as exposure–recovery tests, knockout–rescue tests, and blockage–continuation tests, as well as modulation–response tests, were used to evaluate the essentiality of key events. The essentiality of the MIE (Event 1446: Decrease, Coupling of OXPHOS) is considered high, because direct evidence was collected from multiple specifically designed studies showing simultaneous recovery of OXPHOS coupling (as indicated by the membrane potential) and ATP pool due to removal of the uncouplers (see Weisová et al, 2012), or addition of OXPHOS recoupling agents (see Sibille et al, 1995). The essentiality of KE1 ( Event 1771: Decrease, ATP pool ) is considered high, as supported by direct evidence from a typical inhibition–rescue study showing restimulation of cell proliferation by ATP (Wang et al, 2017) and multiple lines of indirect evidence showing strong positive correlations between ATP and cell proliferation (see Sweet & Singh, 1999).…”