Figure 12. Hsp70 content in CR vs. AL G93A mice. Hsp70 was lower in the quadriceps of CR vs. AL mice (62%, P = 0.002; main effect of diet), and in males vs. females (37%, P = 0.030; main effect of sex). Within each sex, Hsp70 was significantly decreased in CR vs. AL females (64%, P,0.001) and in CR vs. AL males (59%, P = 0.039). 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.g012 In contrast to the effect of CR on increasing MDA, protein oxidation following CR is decreased in G93A mice. We determined that CR significantly decreased PC compared with AL mice. This finding is in accordance with another study using similar energy restriction (60% of AL), whereby CR prevented the age-associated increase in PC in mouse hindlimb skeletal muscle mitochondria [41]. In our study, MDA and PC move in opposing directions as a consequence of CR, which is a finding that proposes some important questions, including whether an increase in PC is concordant with an increase in MDA. A study by Sato el al (1998) revealed that PC levels approximate the unsaturation of lipids in the muscle at opposite extremes, but not in the brain, heart or liver, and although the study did not measure lipid peroxidation, it however reported the peroxidizability and unsaturation indexes (PI and UI, respectively) of each lipid used [42]. Stroke-prone spontaneously hypertensive rats (SHRSP) were fed a diet containing either lard, safflower oil, perilla oil or a fish oil/soybean oil combination [42]. The PI and UI values increased from lard to fish/soybean oil when examining the fatty acid composition of each diet. However, these PI and UI values were altered in vivo depending on the specific tissue analyzed. PC levels in the brain were significantly higher for lard vs. fish/soybean oil only, results inconsistent with the lack of significant difference in the PI or UI in the brain in vivo. In the heart, the fish/soybean oil had a higher PI and UI vs. all other oils, yet PC levels were indistinguishable between the diets. In the liver, the fish/soybean oil group exhibited significantly higher PI and UI vs. all other groups. However, PC content in the liver was higher in the lard diet vs. all other diets, with no difference in PC content between the other oils. In skeletal muscle, fish/soybean oil had significantly higher UI and PI values vs. all other groups, whereas the UI for lard was significantly lower than all other groups. The skeletal muscle PC content in the lard group was significantly lower vs. the safflower, perilla and fish/soybean oil groups, but there was no significant difference in PC content between safflower, perilla and fish/soybean oil groups [42]. These finding indicate that PC and lipid peroxidation levels do not necessarily change in parallel, but are dependent on the tissue and degree of unsaturation. In addition, the variations in the PI and UI of the oils were not reflected proportionately in every tissue. In skeletal muscle, although PC increased in mice fed fish/soybean oil as compared with lard, this increase was similar to mice fed safflower and perilla Caloric Restriction and ALS oils [42]. Hence, even within skeletal muscle, the PI or UI does not dictate tissue PC levels. To further support this, Sen et al (1997) reported that fish oil vs. soy oil supplementation increased lipid peroxidation in the liver, but not in the red gastrocnemius of male Wistar rats [43]. However, fish oil did not induce protein oxidative damage in the liver or red gastrocnemius. There was no correlation between markers of lipid peroxidation and markers of protein oxidation, indicating that PC content is not a reflection of the peroxidizability of lipids in either tissue [43]. Additionally, the n-3 fatty acids in fish oil are highly polyunsaturated and are therefore more readily oxidized, leading to higher lipid peroxidation, as observed in the Sen et al (1997) study. Furthermore, rats co-supplemented with fish oil and vitamin E exhibited significant decreases in both lipid peroxidation and protein oxidation levels in both liver and muscle [43]. Tissue levels of vitamin E in the liver were 20% higher in the rats co-supplemented with fish oil and vitamin E, as opposed to soy oil and vitamin E [43], demonstrating that with a higher intake of highly oxidizable oils, there is a concomitant higher intake of antioxidants. In accordance with Sen et al (1997), Chautan et al (1990) found a 4-fold increase in vitamin E in heart membranes of rats supplemented with PUFAs. Thus, PC content is not only dependent on the PI or UI of the oil, but also on the target tissue (brain, heart, liver and skeletal muscle) and the antioxidant content within the oil or additional antioxidant supplementation. In our diseased animal model, the level of MDA does not reflect the level of PC under CR. It is possible that the negative energy balance as a result of CR had a greater effect on lipids than on protein in the G93A mice. In support of this, Fergani et al (2007) commented on how the defective energy metabolism in female G86R mice, possibly due to skeletal muscle hypermetabolism, leads to increased peripheral use of lipids, signifying a greater reliance on lipids in mSOD1 mouse models [44]. Similarly, short- term fasting in men and women demonstrates a greater reliance on lipids. Basal lipolytic rates during a 14-h fast were increased in women vs. men, but this sexual dimorphism was attenuated at 22- h [45]. Furthermore, female Wistar rats subjected to short-term fasting for 3 or 6 d utilize more fat and maintain lean body mass more efficiently than male rats under the same food restriction [46]. The increase in lipid usage under fasting or food restriction is not only observed in mSOD1 mice, but in humans and non- mSOD1 models as well. Furthermore, the sex difference inherent under energy restriction, with females using more fat vs males, is also conserved in humans and rats. Thus, the sex difference observed in our study, with females having greater levels of lipid peroxidation than males, may be due to the increased lipid usage in female vs. male mSOD1 mice. We ascertained that females had significantly higher levels of MDA in the red gastrocnemius (32%, P = 0.042), but that there were no sex differences in MDA in the white gastrocnemius or in PC levels for either muscle. This finding is interesting since longevity studies in females and males have shown that females have less oxidative stress than males [47,48]. However, female Wistar rats had higher basal levels of H2O2 production, as well as increased GPx activity in liver mitochondria, compared with their male counterparts [49]. In this study, MDA was higher in CR female red gastrocnemius, most likely due to the greater presence of mitochondria in this highly oxidative tissue [50]. In accordance with the increase in MDA levels, there was a substantial upregulation in antioxidant enzymes, MnSOD (red gastrocnemius, 4-fold; white gastrocnemius, 2.2-fold) and Cu/Zn- SOD (red gastrocnemius, 2.6-fold) in CR vs. AL females only. This protective response in females is consistent with studies showing that female rat liver mitochondria exhibit higher antioxidant gene PLoS ONE | www.plosone.org 12 February 2010 | Volume 5 | Issue 2 | e9386