Targeting Mitochondrial Dysfunction with L-Alpha Glycerylphosphorylcholine

Background We hypothesized that L-alpha-glycerylphosphorylcholine (GPC), a deacylatedphosphatidylcholine derivative, can influence the mitochondrial respiratory activity and in this way, may exert tissue protective effects. Methods Rat liver mitochondria were examined with high-resolution respirometry to analyze the effects of GPC on the electron transport chain in normoxic and anoxic conditions. Besides, Sprague-Dawley rats were subjected to sham operation or standardized liver ischemia-reperfusion (IR), with or without GPC administration. The reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG), the tissue myeloperoxidase, xanthine oxidoreductase and NADPH oxidases activities were measured. Tissue malondialdehyde and nitrite/nitrate formation, together with blood superoxide and hydrogen-peroxide production were assessed. Results GPC increased the efficacy of complex I-linked mitochondrial oxygen consumption, with significantly lower in vitro leak respiration. Mechanistically, liver IR injury was accompanied by deteriorated mitochondrial respiration and enhanced ROS production and, as a consequence, by significantly increased inflammatory enzyme activities. GPC administration decreased the inflammatory activation in line with the reduced oxidative and nitrosative stress markers. Conclusion GPC, by preserving the mitochondrial complex I function respiration, reduced the biochemical signs of oxidative stress after an IR episode. This suggests that GPC is a mitochondria-targeted compound that indirectly suppresses the activity of major intracellular superoxide-generating enzymes.


RESULTS
In the SH group, ROS generation with succinate as the sole substrate was strongly inhibited by rotenone and was partly, but not fully, restored by a subsequent addition of antimycin A (S1 Fig.). The ROS generation associated with succinate oxidation in the absence of respiratory chain inhibitors is very sensitive to depolarization of the inner membrane. Thus, ROS release was strongly decreased by the protonophoric uncoupler FCCP. In response to IR, the RET-related ROS production was significantly increased, which supports the notion that the selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia and at least partially responsible for mitochondrial ROS production during reperfusion. When mapping the sites for ROS production at the level of the mitochondrial respiratory chain (i.e. complex I and complex III), it appears that ROS production linked to the reverse electron flux at complex I (succinate alone and rotenone-2 sensitive) is predominant as compared to other sources linked to forward electron flux, since rotenone addition almost fully abolished this ROS production. The lower ROS production in the presence of GPC administration is in agreement with the earlier data (earlier observation in the SUIT protocol -see Figure 2).

EXAMINATION OF COMPLEX I AND II-LINKED RESPIRATION
Additional in vitro experiments were conducted to compare the 30-min anoxia and 30-min reoxygenation (AR) induced changes in complex I and complex II-linked respiration of isolated mitochondria with or without 200 µmol GPC administration (AR and AR+GPC groups respectively; n=8-8). Mitochondria in normoxic environment with or without GPC treatments served as controls (SH and SH+GPC respectively; n=8-8).
Rat liver mitochondria from the left liver lobe were isolated by the method of Gnaiger et al. 1 .
Briefly, mitochondrial pellets were resuspended in isotonic sucrose medium (300 mM sucrose, 0.2 mM EDTA and 10 mM HEPES, adjusted to pH 7.4 with KOH at 4 °C) containing 100 µmol N-acetyl cysteine 2 . For respirometric analysis, isolated mitochondria were suspended in 3 ml MitOx2 medium and weighed into the detection chambers.
During the respirometric analysis, parallel protocols were run in the two chambers for comparison of complex I and complex II-linked oxygen flux. In chamber A, the complex Idependent respiration was measured after the addition of complex II inhibitor malonic acid (10 mM) and 2 mM malate and 10 mM glutamate substrates. Then, saturating concentration of 5 mM ADP for complex I state III respiration was added to the medium. In chamber B, the complex II-linked respiration was determined after the addition of complex I inhibitor 0.5 μM rotenone and 10 mM succinate substrate. Finally, 5 mM ADP was administered for measuring the complex II-dependent oxidative phosphorylation (OxPhos ) capacity.

RESULTS
During the complex I protocol, AR resulted in a significantly lower OxPhos capacity of the mitochondria (complex I-linked state III respiration) in comparison with the SH group. When GPC was administered, however, the respiratory capacity was reversed to the level of SH mitochondria (see S2 Fig.). In contrast, there was no significant difference in the respiratory flux between the groups when the complex II protocol was applied. Thus, neither AR, nor incubation of the respiration medium with GPC affected the function of complex II, as compared with SH mitochondria.
These results suggest that the hypoxic deactivation of complex I might initially act as an intrinsic protective mechanism against the overproduction of ROS and provide a way to recover the cellular bioenergetic function after reoxygenation. However, slower recovery of complex I can contribute to cell injury by both limiting the electron transport required for OxPhos and increasing the production of ROS. Our results demonstrate the sensitivity of complex I-linked OxPhos capacity to AR injury and the protective role of GPC to maintain the electron transport when oxygen concentration rises. The complex II-linked respiration, when investigated separately from the complex I function, was not affected by the AR-related changes.