Mechanism Underlying the Biphasic Effect of ADP on Succinate-Energized Muscle Mitochondrial Respiration

Recently, we reported a novel phenomenon involving basic mitochondrial physiology. When ADP was sequentially added to mouse skeletal muscle mitochondria energized by succinate (in the absence of rotenone, commonly used to block complex I), we found a biphasic (increasing then decreasing) respiratory dose-response to clamped concentrations of ADP. Here we studied the mechanism. First, we confirmed observations that oxaloacetate (OAA) inhibits succinate dehydrogenase (SDH) by assessing the activity of the extracted complex. OAA is known to be difficult to measure by mass spectroscopy. Here we used a nuclear magnetic resonance method to assess OAA as well as malate and fumarate in mitochondria respiring on succinate. When isolated muscle mitochondria were incubated at varying clamped [ADP], we again observed the biphasic response to [ADP], but also found that the decrease in respiration was strongly associated with OAA accumulation. In addition, when a low (0.5 mM) concentration of pyruvate was added to metabolize OAA to citrate, the loss of succinate-supported respiration was reversed. Pyruvate (0.5 mM), added alone (without succinate), was essentially unable to support respiration. The effect of pyruvate to clear OAA and rescue succinate-supported respiration was blocked by inhibition of pyruvate uptake. Further, OAA accumulation with increasing [ADP] corresponded to a decrease in NADH consistent with utilization electrons by the transport system as malate was converted to OAA and consistent with OAA inhibition of SDH. We also found that the dynamic relationship of [ADP] to succinate-energized respiration varies according to muscle fiber type and specific body tissues in addition (as we reported in the past) to being affected in a rodent model of type 2 diabetes.

In summary, the biphasic respiratory response to succinate as respiration progresses from state 4 to 3 is initially triggered by [ADP]-dependent effects on membrane potential followed by inhibition of SDH by OAA.