Mild depolarization of the inner mitochondrial membrane is a crucial component of an anti-aging program

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.

Keywords: aging; antioxidant; mild depolarization; mitochondria; naked mole rat.

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; P_i, inorganic phosphate; Catr, carboxyatractylate.

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). (C) NMR queens (n = 5). (D) NMR subordinates (n = 3).

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.

Fig. 4.

Effect of glucose on the membrane potential of the heart (A and B) and liver (C and D) mitochondria from adult mice or of heart mitochondria from adult NMRs (E and F). In B, D, and F, 1 mM glucose was added before succinate (red curves). Black curves represent absence of glucose. The conditions were the same as described in Fig. 3.

Fig. 5.

(A) H₂O₂ generation by heart mitochondria from adult mice as a function of the membrane potential. The conditions and the effects of various depolarizing agents (malonate, uncouplers [FCCP and SF6847], ADP, glucose, and creatine) are as described in Fig. 3A. (B and C) Membrane potential (B) and H₂O₂ generation (C) as a function of ADP concentration. *P < 0.01; **P < 0.001, for the effects of glucose or creatine.

Fig. 6.

Disappearance of hexokinases bound to skeletal muscle mitochondria during aging of mice. (A) Contents of hexokinases I and II in mitochondria and cytosol. (B) Activities of hexokinases I and II in the mitochondria and cytosol. *P < 0.01; **P < 0.001 (mouse embryos, 12-, 24-, or 30-mo-old mice compared with 3-mo-old mice).

Fig. 7.

(A–J) Aging inhibits glucose- and creatine-stimulated State 4 respiration, as well as the antioxidant activity of these two compounds in mouse skeletal muscle mitochondria. (K) Aging-induced inhibition of glucose phosphorylation in skeletal muscle mitochondria from mice of different ages; comparison of added ADP and ATP. Conditions are as described in Fig. 3.

Fig. 8.

(A and B) Average values of glucose stimulation or the RCR for all tissues in mice and NMRs of various ages. Stimulation of State 4 respiration by glucose disappears with age in mice but not in NMRs. In mice, RCR decreases with age, but this effect is lower than that of glucose stimulation of respiration (A). In NMRs, the RCR does not depend on age (B). RCR was measured as the ratio of the State 3u respiration rate in the presence of uncoupler FCCP to State 4o (respiration rate after oligomycin addition). (C and D) Age-dependent oxidation of lipids and proteins in mouse and NMR tissues. The average values in all tissues of the malondialdehyde level (TBARS probe, lipid peroxidation) (C) and protein carbonylation level (D) are shown. Black, mice; red, NMRs. The data measured for individual tissues are shown in SI Appendix, Figs. S22 and S23.

Fig. 9.

Effect of aging on the expression of hexokinase I (A) and II (B) mRNAs in eight mouse tissues. #P < 0.05; *P < 0.01; **P < 0.001 (12-, 24-, or 30-mo-old mice compared with 3-mo-old mice).

Fig. 10.

Bats (C. perspicillata), similar to NMRs and in contrast to mice, maintain mild polarization for many years. Heart mitochondria were analyzed. Conditions are as detailed in Fig. 3.