Different Effects of Guanine Nucleotides (GDP and GTP) on Protein-Mediated Mitochondrial Proton Leak

Abstract

In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.

Endurance training increases the efficiency of rat skeletal muscle mitochondria

“…To exclude inducible fatty acid-mediated leak through the ATP/ADP anti-porter, UCP-mediated uncoupling was studied in the presence of carboxyatractyloside. Because the inhibition of mitochondrial proton conductance by GTP, but not by GDP, can be considered diagnostic of UCP function [ 40 ], we used GTP to determine UCP activity in isolated skeletal muscle mitochondria from control and trained rats. In both types of mitochondria, with increasing assay temperature, linoleic acid induced greater mitochondrial uncoupling (a greater increase in oxygen uptake and a greater decrease in mΔΨ), while GTP caused a more pronounced inhibition of this uncoupling (a greater decrease in the oxidation rate and a greater increase in mΔΨ), indicating an elevation of UCP-mediated uncoupling with increasing assay temperature.…”

Ketosis may promote brain macroautophagy by activating Sirt1 and hypoxia-inducible factor-1

“…However, the increased efficiency of respiratory electron flow in ketoneconsuming cells is not entirely a function of increased uncoupling protein expression, as it is observed in isolated mitochondria incubated with ketone bodies; concurrently, mitochondrial generation of superoxide decreases [12]. Conceivably, by promoting conversion of succinyl-CoA to succinate without concurrent generation of GTP, mitochondrial ketone metabolism might increase the function of GTP-inhibitable uncoupling proteins [15]. Systemically, an increase in respiratory uncoupling has been observed during ketosis [16].…”

Cell Death and Heart Failure in Obesity: Role of Uncoupling Proteins

“…UCP2 and UCP3 are negatively regulated by purine nucleotides such as GTP, ATP, and GDP which are considered as physiological inhibitors [ 29 ]. Nevertheless FFAs were suggested as a direct activators of UCPs that increase conductance and dissipate H + electrochemical gradient into heat.…”