Effect of aging, caloric restriction, and uncoupling protein 3 (UCP3) on mitochondrial proton leak in mice

Abstract

Mitochondrial proton leak may modulate reactive oxygen species (ROS) production and play a role in aging. The purpose of this study was to determine proton leak across the life span in skeletal mitochondria from calorie-restricted and UCP2/3 overexpressing mice. Proton leak in isolated mitochondria and markers of oxidative stress in whole tissue were measured in female C57BL/6J mice fed ad-libitum (WT-Control) or a 30% calorie-restricted (WT-CR) diet, and in mice overexpressing UCP2 and UCP3 (Positive-TG), their non-overexpressing littermates (Negative-TG) and UCP3 knockout mice (UCP3KO). Proton leak in WT-CR mice was lower than that of control mice at 8 and 26 months of age. The Positive-TG mice had greater proton leak than the Negative-TG and UCP3KO mice at 8 months of age, but this difference disappeared by 19 and 26 months. Lipid peroxidation was generally lower in WT-CR vs. WT-Control mice and UCP3KO mice had greater concentrations of T-BARS (thiobarbituric acid reactive substances, a measure of lipid peroxidation) than did Positive-TG and Negative-TG. The results of this study indicate that sustained increases in muscle mitochondrial proton leak are not responsible for alterations in life span with calorie restriction or UCP3 overexpression in mice. However, UCP3 may contribute to the actions of CR through mechanisms distinct from increasing basal proton leak.

Figure 1

Proton leak kinetics in muscle mitochondria isolated from mice that overexpress UCP2/3 (Positive-TG), the non-overexpressing littermates of these mice (Negative-TG) and mice that do not have the gene for UCP3 (UCP3KO) at 8- (A), 19- (B), and 26- (C) months of age. The furthermost point on the right represents maximal leak-dependent oxygen consumption and was determined by addition of saturating amounts of oligomycin. Kinetic responses of proton leak block were determined by incremental additions of malonate (0.16–1.25 mM) to the mitochondria at State 4. Each point represents the mean ± SE; n = 8 mice/group.

Figure 2

Proton leak kinetics in muscle mitochondria isolated from mice fed ad-libitum (WT-Control) or calorie restricted to 70% of ad-libitum fed mice (WT-CR) at 8- (A), 19- (B), and 26- (C) months of age. The furthermost point on the right represents maximal leak-dependent oxygen consumption and determined by addition of saturating amounts of oligomycin. Kinetic response of proton leak block was determined by incremental additions of malonate (0.16–1.25 mM) to the mitochondria at State 4. Each point represents the mean ± SE; n = 8 mice/group.

Figure 3

Proton leak kinetics in muscle mitochondria isolated from mice that have been calorie restricted to 70% of ad-libitum fed mice at 8-, 19-, 26- and 30-months of age. The furthermost point on the right represents maximal leak-dependent oxygen consumption and determined by addition of saturating amounts of oligomycin. Kinetic response of proton leak block was determined by incremental additions of malonate (0.16–1.25 mM) to the mitochondria at State 4. Each point represents the mean ±SE; n = 8 mice/group.

Figure 4

T-BARSs, and protein carbonyl concentration in lower hind limb muscle of gene-manipulated mice, ad-libitum fed and calorie restricted C57BL/6J mice. Within a strain (C57BL/6J or Genetically Altered) and group (WT-Control or WT-CR; Positive-TG, Negative-TG, or UCP3KO), bars sharing or not having an upper case letter do not differ significantly (i.e., age differences). Within a strain (C57BL/6J or Genetically Altered) and age, bars sharing or not having an lower case letter do not differ significantly (i.e., group difference). MDA, malonidaldehyde. Positive-TG, overexpression of UCP2/3; Negative-TG, non-overxpression littermates of Positive-TG; UCP3KO, UCP3 knockout mice; WT-Control, ad libitum fed C57BL/6J; WT-CR, calorie restricted C57BL/6J.