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O P E N A C C E S S S O U R C E : PNAS
Significance
The mitochondria, organelles that produce the largest amounts of ATP and reactive oxygen species (mROS) in living cells, are equipped with a universal mechanism that can completely prevent mROS production. This mechanism consists of mild depolarization of the inner mitochondrial membrane to decrease the membrane potential to a level sufficient to form ATP but insufficient to generate mROS. In short-lived mice, aging is accompanied by inactivation of the mild depolarization mechanism, resulting in chronic poisoning of the organism with mROS. However, mild depolarization still functions for many years in long-lived naked mole rats and bats.
Abstract
The mitochondria of various tissues from mice, naked mole rats (NMRs), and bats possess two mechanistically similar systems to prevent the generation of mitochondrial reactive oxygen species (mROS): hexokinases I and II and creatine kinase bound to mitochondrial membranes. Both systems operate in a manner such that one of the kinase substrates (mitochondrial ATP) is electrophoretically transported by the ATP/ADP antiporter to the catalytic site of bound hexokinase or bound creatine kinase without ATP dilution in the cytosol. One of the kinase reaction products, ADP, is transported back to the mitochondrial matrix via the antiporter, again through an electrophoretic process without cytosol dilution. The system in question continuously supports H+-ATP synthase with ADP until glucose or creatine is available. Under these conditions, the membrane potential, ∆ψ, is maintained at a lower than maximal level (i.e., mild depolarization of mitochondria). This ∆ψ decrease is sufficient to completely inhibit mROS generation. In 2.5-y-old mice, mild depolarization disappears in the skeletal muscles, diaphragm, heart, spleen, and brain and partially in the lung and kidney. This age-dependent decrease in the levels of bound kinases is not observed in NMRs and bats for many years. As a result, ROS-mediated protein damage, which is substantial during the aging of short-lived mice, is stabilized at low levels during the aging of long-lived NMRs and bats. It is suggested that this mitochondrial mild depolarization is a crucial component of the mitochondrial anti-aging system.
In 1997, one of the authors of this paper (V.P.S.) and his coworkers S. Korshunov and A. Starkov reported that a rather small decrease in the mitochondrial electric potential results in the complete arrest of the generation of mitochondrial reactive oxygen species (mROS) (1). In particular, the activation of oxidative phosphorylation by the addition of ADP to isolated mitochondria decreased the membrane potential by ∼20%, which is sufficient to prevent H2O2 formation by these organelles. In 2004 to 2008, this effect was investigated by A. Galina and colleagues in Rio de Janeiro (2⇓–4). The authors described a novel antioxidant mechanism in mitochondria from the rat brain. This mechanism consists of the cyclic movement of ADP produced by hexokinase or creatine kinase bound to the outer surface of the outer or inner mitochondrial membrane, respectively. Researchers proposed that ATP (formed inside mitochondria from ADP and phosphate Pi at the expense of the respiratory chain-produced protonic potential, Δμ¯¯H+), is used to regenerate ADP in the kinase active sites. As a result, glucose-6-phosphate (G6P) or creatine phosphate are formed. ADP produced by these kinases returns to the matrix (without dilution by the cytosol) to be converted again into ATP (schemes shown in Fig. 1 and SI Appendix, Fig. S2).
Fig. 1 . Interrelations of respiratory chain Complexes I, II, III, and IV; H+-ATP-synthase (Complex V); ∆ψ; ATP/ADP-antiporter; porin; mitochondrion-bound hexokinase I or II; and mROS. Complex I, NADH-CoQ oxidoreductase; Complex II, succinate dehydrogenase; Complex III, CoQH2-cytochrome c oxidoreductase; Complex IV, cytochrome c oxidase; Pi, inorganic phosphate; Catr, carboxyatractylate.
The difference in the electrical potentials of the mitochondrial membrane, Δψ, is the main component of mitochondrial Δμ¯¯H+. The activation of two kinase-mediated cycles results in a decrease in Δψ. This depolarization is rather small, however, since ATP synthesis from ADP and Pi becomes impossible if Δψ (initially ∼190 mV) decreases to ∼140 mV (1). Therefore, the kinase-induced effect is termed mild depolarization. The situation resembles the function of uncoupling protein 1 (UCP1), which serves as a protonophore if Δψ is greater than a critical value (5). The kinase-linked mild depolarization appears to be a more sophisticated mechanism than UCP1, since it is accompanied by some useful bioenergetic functions (rather than the complete dissipation of respiration energy as heat by UCP1). In the case of hexokinase or creatine kinase, the mobilization of carbohydrate catabolism (glucose phosphorylation to G6P) or the accumulation of a high-energy buffer (creatine phosphate) occurs. G6P is the first intermediate in a complicated metabolic pathway resulting in the formation of pyruvate, which is used in respiration during the Krebs cycle or, alternatively, converted to lactate, the final product of glycolysis.
In tissues other than the brain, the mild depolarization mechanism, according to Galina et al. (2⇓–4), is much less active (kidney), almost inactive (heart), or absent (liver). In this paper, we summarize our analysis of the role of mitochondria-bound kinases in preventing mROS generation. Adult mice and mouse embryos, naked mole rats (NMRs) and their embryos, and bats of different ages were investigated. We found that in adult mice, all tissues but the liver are equipped with bound kinases that can completely prevent mROS formation. In mouse embryos, mild depolarization was observed in all tissues studied, including the liver. In our experiments, all tissues in NMRs exhibited a mild depolarization mechanism.
We also observed that aging in mice up to age 2.5 y is accompanied by partial or even complete arrest of the mild depolarization phenomenon in different tissues. This arrest does not occur in NMRs and bats at least before age 12 to 13 y.
We assume that the slow permanent attenuation of mild depolarization in mice represents a result of the operation of the aging program, an effect that increases mROS production by the respiratory chain. Regarding mild depolarization, it seems to be a novel, universal anti-aging mechanism that specifically prevents the pro-aging effect of mROS.
Results
Mild Depolarization Is Inherent in Different Tissues.
In the first series of experiments, we measured the amounts of soluble and mitochondria-bound hexokinases I and II in eight tissues from adult mice and NMRs of different ages, as well as in six tissues from mouse embryos (Fig. 2 and SI Appendix, Fig. S3). In adult mice, consistent with the data reported by Galina et al. (2⇓–4), very high concentrations of bound hexokinases were detected in the brain and very low levels were detected in the liver. However, surprisingly, rather high hexokinase levels were also observed in the heart, kidney, diaphragm muscle, skeletal muscle, lung, and spleen. In mouse embryos and adult NMRs, measurable amounts of bound hexokinases were present in all tissues studied. Significant differences between NMR queens and NMR subordinates were not observed for most tissues (Fig. 2 C and D); moreover, in all tissues from adult mice except skeletal muscle, bound hexokinase I dominated over bound hexokinase II which is known to have much lower affinity to glucose. In skeletal muscle tissue from 3-mo-old mice, hexokinase II strongly dominated over hexokinase I (Fig. 2A). Levels of the two hexokinases were equal in skeletal muscle tissue from 3-mo-old NMRs, but hexokinase II was higher in 3-y-old and older animals (SI Appendix, Fig. S11). The late appearance of hexokinase II dominance in skeletal muscles is apparently a trait of neoteny (prolongation of youth), explaining the unique longevity of NMRs (6⇓–8).
Fig. 2 . Contents of mitochondria-bound and soluble hexokinases (HK) I and II in different tissues from mice and NMRs. Western blot analyses were performed with the appropriate monoclonal antibodies. Mean values for three to four repeats are presented. (A) 3-mo-old mice (n = 7). (B) Mouse embryos (n = 21). © NMR queens (n = 5). (D) NMR subordinates (n = 3).
Confocal microscopy of cultures of mouse and NMR liver fibroblasts revealed that hexokinase is bound to mitochondria in intact cells (SI Appendix, Fig. S4). A subsequent series of experiments (Fig. 3) was carried out in mitochondria isolated from various tissues of mice and NMRs. Respiration and H2O2 generation were measured (Fig. 3 A, C, E, and G–J and Fig. 3 B, D, F, and K, respectively). The respiration rate was followed polarographically when [O2] was estimated as a function of time. Respiration was initiated by the addition of a respiratory substrate, succinate (succ). Before succinate addition (e.g., black curve in Fig. 3A), O2 consumption was negligible, since respiration is limited by the absence of its substrate (State 1). Succinate slightly stimulated respiration; now it was limited by the absence of ADP (energy acceptor, State 2). The addition of ADP increased the respiration rate to the maximal level (State 3); however, within a few minutes, O2 consumption was lowered to the level before ADP addition, since ADP was exhausted while being phosphorylated to ATP (State 4). Carboxyatractylate (CAtr), the ATP/ADP antiporter inhibitor, and oligomycin, a H+-ATP synthase inhibitor, decreased O2 consumption when added before ADP. The maximal O2 consumption rate was achieved by adding an uncoupler, carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a protonophorous uncoupler of respiration and phosphorylation (9). The red curves in Fig. 3 show mitochondrial respiration when glucose or creatine was added in State 2. It can be seen that this addition does not influence States 2 and 3, but State 4 does not exist now. In fact, State 3 continues as if the added ADP was not exhausted. The novel state was inhibited by CAtr, and the inhibition was abolished by FCCP. Fig. 3B shows the formation of mitochondrial H2O2 measured by fluorescent spectrophotometry (using Amplex Red reagent and horseradish peroxidase). The inhibition of respiration at the State 3 to State 4 transition due to ADP exhaustion was accompanied by a strong increase in H2O2 formation (black curve). This increase was completely abolished by adding glucose (red curve).
Fig. 3 . Respiration and H2O2 generation by mitochondria isolated from different tissues from adult mice (A–F), mouse embryos (G–I), and adult NMRs (J and K). Additions to the incubation mixture: 50 μM CArt, 3 mM creatine, 1 μg oligomycin (Oligo), 1 mM glucose, 10−7 M FCCP, 2 μM rotenone, and 0.5 mM KCN. Mitochondria were isolated in 300 mM mannitol, 0.5 mM EDTA, and 20 mM Hepes-KOH, pH 7.6. Mitochondria were incubated in medium containing 220 mM mannitol, 10 mM potassium lactobionate, 5 mM potassium phosphate, 2 mM MgCl2, 10 μM EGTA, and 20 mM Hepes-KOH, pH 7.6. State 2 respiration was initiated by adding succinate (10 mM). State 3 or 3u was induced by the addition of 100 µM ADP or 10 nM FCCP, respectively. The concentration of mitochondria in the incubation vessel was 0.1 mg protein/mL. The temperature was 25 °C.
The data in Fig. 3 A and B were obtained using isolated mouse brain mitochondria, confirming the results of Galina et al. (2, 4). Further experiments revealed that many mouse tissues other than the brain demonstrate complete arrest of State 4 H2O2 formation by adding glucose (Figs. 3 C and D and SI Appendix, Figs. S6 and S12). On the other hand, in line with the observations of Galina et al., mouse liver mitochondria do not respond to the glucose addition with an increase in respiration (Fig. 3 E and F). However, the mouse embryo liver showed an effect of glucose similar to that seen in other tissues (Fig. 3H).
Consistent with observations in the brain reported by Galina et al. (3), creatine replaced sugars in stimulating the State 4 respiration of the mouse mitochondria (Fig. 3H). Similar to glucose, the effect of creatine was completely blocked by oligomycin and CAtr (Fig. 3H). Creatine-induced respiration was specifically inhibited by the creatine kinase inhibitor guanidopropionic acid (SI Appendix, Fig. S5D). The glucose effect was strongly suppressed by the hexokinase inhibitor glucose 6-phosphate (G6P) (Figs. 7 and 10 and SI Appendix, Figs. S5, S9, and S10) but not by fructose 6-phosphate (F6P) and 2-deoxyglucose 6-phosphate (DOG6P), which cannot inhibit hexokinase (SI Appendix, Fig. S5).
Fig. 4 shows the generation of Δψ by heart or liver mitochondria from adult mice and NMRs. The addition of ADP to heart mitochondria induced a decrease in Δψ (mild depolarization of the mitochondrial membrane due to the State 2 to State 3 transition). When ADP was exhausted (the State 3 to State 4 transition), Δψ increased. This increase disappeared on the addition of 1 mM glucose. Subsequent additions of the uncoupler FCCP completely depolarized the mitochondria. In the adult mouse liver, glucose did not affect Δψ. In mouse embryos and adult NMRs, a glucose effect was observed in both the liver and the heart.
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Edited by Engadin, 14 March 2020 - 09:07 PM.
