Imbalance between proliferation and cell death accounts for several age-linked diseases. Aging, calorie restriction (CR), and fat source are all factors that may influence apoptotic signaling in liver, an organ that plays a central metabolic role in the organism. Here, we have studied the combined effect of these factors on a number of apoptosis regulators and effectors. For this purpose, animals were fed diets containing different fat sources (lard, soybean oil, or fish oil) under CR for 6 or 18 months. An age-linked increase in the mitochondrial apoptotic pathway was detected with CR, including a decrease in Bcl-2/Bax ratio, an enhanced release of cytochrome c to the cytosol and higher caspase-9 activity. However, these changes were not fully transmitted to the effectors apoptosis-inducing factor and caspase-3. CR (which abated aging-related inflammatory responses) and dietary fat altered the activities of caspases-8, -9, and -3. Apoptotic index (DNA fragmentation) and mean nuclear area were increased in aged animals with the exception of calorie-restricted mice fed a lard-based fat source. These results suggest possible protective changes in hepatic homeostasis with aging in the calorie-restricted lard group.

Calorie restriction (CR) has been repeatedly shown to prevent cancer, diabetes, hypertension, and other age-related diseases in a wide range of animals, including non-human primates and humans. In rodents, CR also increases lifespan and is a powerful tool for studying the aging process. Recently, it has been reported in mice that dietary fat plays an important role in determining lifespan extension with 40% CR. In these conditions, animals fed lard as dietary fat showed an increased longevity compared with mice fed soybean or fish oils. In this paper, we study the effect of these dietary fats on structural and physiological parameters of kidney from mice maintained on 40% CR for 6 and 18 months. Analyses were performed using quantitative electron microcopy techniques and protein expression in Western blots. CR mitigated most of the analyzed age-related parameters in kidney, such as glomerular basement membrane thickness, mitochondrial mass in convoluted proximal tubules and autophagic markers in renal homogenates. The lard group showed improved preservation of several renal structures with aging when compared to the other CR diet groups. These results indicate that dietary fat modulates renal structure and function in CR mice and plays an essential role in the determination of health span in rodents.

Loss of skeletal muscle mass and function is a hallmark of aging. This phenomenon has been related to a dysregulation of mitochondrial function and proteostasis. Calorie restriction (CR) has been demonstrated to delay aging and preserve function until late in life, particularly in muscle. Recently, we reported the type of dietary fat plays an important role in determining life span extension with 40% CR in male mice. In these conditions, lard fed mice showed an increased longevity compared to mice fed soybean or fish oils. In this article, we analyze the effect of 40% CR on muscle mitochondrial mass, autophagy, and mitochondrial dynamics markers in mice fed these diets. In CR fed animals, lard preserved muscle fibers structure, mitochondrial ultrastructure, and fission/fusion dynamics and autophagy, not only compared to control animals, but also compared with CR mice fed soybean and fish oils as dietary fat. We focus our discussion on dietary fatty acid saturation degree as an essential predictor of life span extension in CR mice.

To investigate the role mitochondrial membrane lipids play in the actions of CR (calorie restriction), C57BL/6 mice were assigned to four groups (control and three 40% CR groups) and the CR groups were fed diets containing soya bean oil (also in the control diet), fish oil or lard. The fatty acid composition of the major mitochondrial phospholipid classes, proton leak and H(2)O(2) production were measured in liver mitochondria following 1 month of CR. The results indicate that mitochondrial phospholipid fatty acids reflect the PUFA (polyunsaturated fatty acid) profile of the dietary lipid sources. CR significantly decreased the capacity of ROS (reactive oxygen species) production by Complex III but did not markedly alter proton leak and ETC (electron transport chain) enzyme activities. Within the CR regimens, the CR-fish group had decreased ROS production by both Complexes I and III, and increased proton leak when compared with the other CR groups. The CR-lard group showed the lowest proton leak compared with the other CR groups. The ETC enzyme activity measurements in the CR regimens showed that Complex I activity was decreased in both the CR-fish and CR-lard groups. Moreover, the CR-fish group also had lower Complex II activity compared with the other CR groups. These results indicate that dietary lipid composition does influence liver mitochondrial phospholipid composition, ROS production, proton leak and ETC enzyme activities in CR animals.

Calorie restriction (CR) without malnutrition extends life span in several animal models. It has been proposed that a decrease in the amount of polyunsaturated fatty acids (PUFAs), and especially n-3 fatty acids, in membrane phospholipids may contribute to life span extension with CR. Phospholipid PUFAs are sensitive to dietary fatty acid composition, and thus, the purpose of this study was to determine the influence of dietary lipids on life span in CR mice. C57BL/6J mice were assigned to four groups (a 5% CR control group and three 40% CR groups) and fed diets with soybean oil (high in n-6 PUFAs), fish oil (high in n-3 PUFAs), or lard (high in saturated and monounsaturated fatty acids) as the primary lipid source. Life span was increased (p < .05) in all CR groups compared to the Control mice. Life span was also increased (p < .05) in the CR lard mice compared to animals consuming either the CR fish or soybean oil diets. These results indicate that dietary lipid composition can influence life span in mice on CR, and suggest that a diet containing a low proportion of PUFAs and high proportion of monounsaturated and saturated fats may maximize life span in animals maintained on CR.

In this paper we analyzed changes in hepatocyte mitochondrial mass and ultrastructure as well as in mitochondrial markers of fission/fusion and biogenesis in mice subjected to 40% calorie restriction (CR) for 18 months versus ad libitum-fed controls. Animals subjected to CR were separated into three groups with different dietary fats: soybean oil (also in controls), fish oil and lard. Therefore, the effect of the dietary fat under CR was studied as well. Our results show that CR induced changes in hepatocyte and mitochondrial size, in the volume fraction occupied by mitochondria, and in the number of mitochondria per hepatocyte. Also, mean number of mitochondrial cristae and lengths were significantly higher in all CR groups compared with controls. Finally, CR had no remarkable effects on the expression levels of fission and fusion protein markers. However, considerable differences in many of these parameters were found when comparing the CR groups, supporting the idea that dietary fat plays a relevant role in the modulation of CR effects in aged mice.

The polyunsaturated nature of n-3 fatty acids makes them prone to oxidative damage. However, it is not clear if n-3 fatty acids are simply a passive site for oxidative attack or if they also modulate mitochondrial reactive oxygen species (ROS) production. The present study used fat-1 transgenic mice, that are capable of synthesizing n-3 fatty acids, to investigate the influence of increases in n-3 fatty acids and resultant decreases in the n-6:n-3 ratio on liver mitochondrial H(2)O(2) production and electron transport chain (ETC) activity. There was an increase in n-3 fatty acids and a decrease in the n-6:n-3 ratio in liver mitochondria from the fat-1 compared to control mice. This change was largely due to alterations in the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine, with only a small percentage of fatty acids in cardiolipin being altered in the fat-1 animals. The lipid changes in the fat-1 mice were associated with a decrease (p<0.05) in the activity of ETC complex I and increases (p<0.05) in the activities of complexes III and IV. Mitochondrial H(2)O(2) production with either succinate or succinate/glutamate/malate substrates was also decreased (p<0.05) in the fat-1 mice. This change in H(2)O(2) production was due to a decrease in ROS production from ETC complex I in the fat-1 animals. These results indicate that the fatty acid changes in fat-1 liver mitochondria may at least partially oppose oxidative stress by limiting ROS production from ETC complex I.

The Membrane Theory of Aging proposes that lifespan is inversely related to the level of unsaturation in membrane phospholipids. Calorie restriction (CR) without malnutrition extends lifespan in many model organisms, which may be related to alterations in membrane phospholipids fatty acids. During the last few years our research focused on studying how altering the predominant fat source affects the outcome of CR in mice. We have established four dietary groups: one control group fed 95 % of a pre-determined ad libitum intake (in order to prevent obesity), and three CR groups fed 40 % less than ad libitum intake. Lipid source for the control and one of the CR groups was soybean oil (high in n-6 PUFA) whereas the two remaining CR groups were fed diets containing fish oil (high in n-3 PUFA), or lard (high in saturated and monounsaturated fatty acids). Dietary intervention periods ranged from 1 to 18 months. We performed a longitudinal lifespan study and a cross-sectional study set up to evaluate several mitochondrial parameters which included fatty acid composition, H(+) leak, activities of electron transport chain enzymes, ROS generation, lipid peroxidation, mitochondrial ultrastructure, and mitochondrial apoptotic signaling in liver and skeletal muscle. These approaches applied to different cohorts of mice have independently indicated that lard as a fat source often maximizes the effects of 40 % CR on mice. These effects could be due to significant increases of monounsaturated fatty acids levels, in accordance with the Membrane Theory of Aging.