Figure 8. Potein oxidation in CR vs. AL G93A mice. (A) PC was lower in CR vs. AL red gastrocnemius (62%, P,0.001; main effect of diet). Within each sex, PC was lower in CR vs. AL females (50%, P = 0.012), and in CR vs. AL males (74%, P,0.001). (B) PC was lower in CR vs. AL white gastrocnemius (30%, P = 0.003; main effect of diet). PC was lower in CR vs. AL females (43%, P,0.001). Data are presented as means 6 SEM. n = 27; AL, 7 males and 7 females; CR, 7 males and 6 females. Asterisks denote significant changes (P#0.05 vs. AL). doi:10.1371/journal.pone.0009386.g008 evidence for the negative implications of reduced energy intake in the pathogenesis of disease [25], specifically that long-term CR hastens the clinical onset of disease, disease progression and life span in G93A mice [9,10]. However, the molecular basis of the negative effects of CR on G93A mouse lifespan and survival remained undetermined. In the present study, we investigated the effect of CR on markers of mitochondrial bioenergetics, oxidative stress, antioxidant enzyme capacity, inflammation, stress response and apoptosis. CR induces mitochondrial biogenesis and bioenergetic efficien- cy in both animals and humans [26–28]. In the current study, we observed that CR significantly increased the ratio of COX-IV to citrate synthase protein content in male G93A mice. However, this increase did not translate into enhanced mitochondrial oxidative capacity since the ratio of COX to citrate synthase activity (marker of mitochondrial bioenergetic efficiency) is reduced in CR male G93A mice. We propose that CR increases the protein content of subunits of mitochondrial repiratory chain in male G93A mice to compensate for reduced mitochondrial bioenergetic efficiency. Interestingly, patients with primary mitochondrial myopathy as a result of point mutation in their mitochondrial genome show similar increases in mitochondrial content without an adaptive increase in mitochondrial function [29–31]. The discordant effects Caloric Restriction and ALS of CR observed on mitochondrial content vs. function suggest that the mitochondrial genome may have been compromised due to higher levels of oxidative stress in G93A mice as previously reported [31–34], and that an increase in mitochondrial biogenesis in response to CR [25–27] only results in accumulation of dysfunctional mitochondrial proteins. Furthermore, it has been shown that 95 d old G93A mice have significantly elevated levels of MDA (marker of lipid peroxida- tion) and PC (markers of protein oxidation) in skeletal muscle, as well as a substantial increase in MnSOD, Cu/Zn-SOD and catalase enzyme activity compared with wild-type mice [32]. Thus, G93A mice have a basal increase in oxidative stress due to the overexpression of its mSOD1 protein [32–35]. In the present study, we determined that CR significantly increased MDA in both the red and white gastrocnemius. It is possible that decreases in mitochondrial oxidant production, as a result of long-term CR, failed to balance the increase in free radical production from the mutant enzyme found mainly in the cytosol, leading to a net increase in oxidative stress [9]. One short-term CR study showed a similar pattern, with significantly higher lipid peroxidation potential in the hepatic microsomes of slightly restricted (75% of AL) young (3.5 mo) mice compared with severely restricted (50% of AL) young mice, slightly and severely restricted old (27 mo) mice, and severely restricted senescent (45 mo) mice [36]. Lipid peroxidation potential, induced by the addition of Fe2+, was determined by the presence of lipid hydroperoxides (LOOH) in the microsomes [36]. After the addition of increasing concentrations of Fe2+, the severely restricted young mice and the slightly and severely restricted old mice exhibited the lowest lipid peroxidation potential, whereas the lipid peroxidation potential in the severely restricted senescent mice increased to intermediate levels (approximately 60% lower vs. the slightly restricted young) [36]. Surprisingly, the slightly restricted young mice exhibited the highest lipid peroxidation potential. These results indicate that severe short- term CR in the young and old mice lowered lipid peroxidation potential, but in the senescent mice this effect was attenuated as a result of age. Thus, the magnitude of energy restriction, as well as age, have an impact on the peroxidative potential of hepatic microsomes [36]. The authors suggest that severe CR has a greater effect in reducing lipid peroxidation earlier in life [36], hence the higher peroxidative potential in the young slightly restricted mice. In confirmation, a previous study found that dietary restriction of 55 kcal/wk significantly reduced lipid peroxidation in 12-month old mouse liver, but not in 24-month old restricted mice [37]. Therefore, the CR-induced reduction in lipid peroxidation can be attenuated by age. Short-term CR (2 mo; ,60% of AL) in male Fisher 344 rats showed decreased H2O2 production (14% reduction) and a diminution in mitochondrial antioxidant enzyme activities (SOD2, 39% reduction; GPx, 19% reduction), but increased PC in cardiac muscle as compared with AL rats [38]. Judge et al (2003) postulated that the increase in PC may be a result of the reduction in SOD2 and GPx activity and/or that protein oxidation is transiently elevated in cardiac muscle [38]. Further- more, CR (2 mo; 70% of AL) increased MDA by 3-fold in the liver and by 50% in the heart of male Sprague-Dawley rats, an animal model with elevated basal levels of stress-induced corticosterone, vs. AL-fed rats [39]. Conversely, Gredilla et al (2001) established that 6 weeks of CR (60% of AL) significantly decreased mitochondrial and nuclear marker of DNA damage, 8-OHdG, and H2O2 production in the liver of male Wistar rats [40]. The elevation we observed in MDA in the skeletal muscle of G93A mice, which exhibit increased basal levels of oxidative stress [32], is PLoS ONE | www.plosone.org 10 February 2010 | Volume 5 | Issue 2 | e9386