Pilar Caro

Methionine restriction decreases mitochondrial oxygen radical generation and leak as well as oxidative damage to mitochondrial DNA and proteins

Previous studies have consistently shown that caloric restriction (CR) decreases mitochondrial reactive oxygen species (ROS) (mitROS) generation and oxidative damage to mtDNA and mitochondrial proteins, and increases maximum longevity, although the mechanisms responsible for this are unknown. We recently found that protein restriction (PR) also produces these changes independent of energy restriction. Various facts link methionine to aging, and methionine restriction (MetR) without energy restriction increases, like CR, maximum longevity. We have thus hypothesized that MetR is responsible for the decrease in mitROS generation and oxidative stress in PR and CR. In this investigation we subjected male rats to exactly the same dietary protocol of MetR that is known to increase their longevity. We have found, for the first time, that MetR profoundly decreases mitROS production, decreases oxidative damage to mtDNA, lowers membrane unsaturation, and decreases all five markers of protein oxidation measured in rat heart and liver mitochondria. The concentration of complexes I and IV also decreases in MetR. The decrease in mitROS generation occurs in complexes I and III in liver and in complex I in heart mitochondria, and is due to an increase in efficiency of the respiratory chain in avoiding electron leak to oxygen. These changes are strikingly similar to those observed in CR and PR, suggesting that the decrease in methionine ingestion is responsible for the decrease in mitochondrial ROS production and oxidative stress, and possibly part of the decrease in aging rate, occurring during caloric restriction.—Sanz, A., Caro, P., Ayala, V., Portero‐Otin, M., Pamplona, R., Barja, G. Methionine restriction decreases mitochondrial oxygen radical generation and leak as well as oxidative damage to mitochondrial DNA and proteins. FASEB J. 20, 1064–1073 (2006)

Forty percent and eighty percent methionine restriction decrease mitochondrial ROS generation and oxidative stress in rat liver

Dietary restriction (DR) lowers mitochondrial reactive oxygen species (ROS) generation and oxidative damage and increases maximum longevity in rodents. Protein restriction (PR) or methionine restriction (MetR), but not lipid or carbohydrate restriction, also cause those kinds of changes. However, previous experiments of MetR were performed only at 80% MetR, and substituting dietary methionine with glutamate in the diet. In order to clarify if MetR can be responsible for the lowered ROS production and oxidative stress induced by standard (40%) DR, Wistar rats were subjected to 40% or 80% MetR without changing other dietary components. It was found that both 40% and 80% MetR decrease mitochondrial ROS generation and percent free radical leak in rat liver mitochondria, similarly to what has been previously observed in 40% PR and 40% DR. The concentration of complexes I and III, apoptosis inducing factor, oxidative damage to mitochondrial DNA, five different markers of protein oxidation, glycoxidation or lipoxidation and fatty acid unsaturation were also lowered. The results show that 40% isocaloric MetR is enough to decrease ROS production and oxidative stress in rat liver. This suggests that the lowered intake of methionine is responsible for the decrease in oxidative stress observed in DR.

Forty Percent Methionine Restriction Decreases Mitochondrial Oxygen Radical Production and Leak at Complex I During Forward Electron Flow and Lowers Oxidative Damage to Proteins and Mitochondrial DNA in Rat Kidney and Brain Mitochondria

Eighty percent dietary methionine restriction (MetR) in rodents (without calorie restriction), like dietary restriction (DR), increases maximum longevity and strongly decreases mitochondrial reactive oxygen species (ROS) production and oxidative stress. Eighty percent MetR also lowers the degree of membrane fatty acid unsaturation in rat liver. Mitochondrial ROS generation and the degree of fatty acid unsaturation are the only two known factors linking oxidative stress with longevity in vertebrates. However, it is unknown whether 40% MetR, the relevant methionine restriction degree to clarify the mechanisms of action of standard (40%) DR can reproduce these effects in mitochondria from vital tissues of strong relevance for aging. Here we study the effect of 40% MetR on ROS production and oxidative stress in rat brain and kidney mitochondria. Male Wistar rats were fed during 7 weeks semipurified diets differing only in their methionine content: control or 40% MetR diets. It was found that 40% MetR decreases mitochondrial ROS production and percent free radical leak (by 62-71%) at complex I during forward (but not during reverse) electron flow in both brain and kidney mitochondria, increases the oxidative phosphorylation capacity of brain mitochondria, lowers oxidative damage to kidney mitochondrial DNA, and decreases specific markers of mitochondrial protein oxidation, lipoxidation, and glycoxidation in both tissues. Forty percent MetR also decreased the amount of respiratory complexes I, III, and IV and apoptosis-inducing factor (AIF) in brain mitochondria and complex IV in kidney mitochondria, without changing the degree of mitochondrial membrane fatty acid unsaturation. Forty percent MetR, differing from 80% MetR, did not inhibit the increase in rat body weight. These changes are very similar to the ones previously found during dietary and protein restriction in rats. We conclude that methionine is the only dietary factor responsible for the decrease in mitochondrial ROS production and oxidative stress, and likely for part of the longevity extension effect, occurring in DR.

Effect of Lipid Restriction on Mitochondrial Free Radical Production and Oxidative DNA Damage

Abstract:  Many studies have shown that caloric restriction (40%) decreases mitochondrial reactive oxygen species (ROS) generation in rodents . Moreover, we have recently found that 7 weeks of 40% protein restriction without strong caloric restriction also decreases ROS production in rat liver. This is interesting since it has been reported that protein restriction can also extend longevity in rodents. In the present study we have investigated the possible role of dietary lipids in the effects of caloric restriction on mitochondrial oxidative stress. Using semipurified diets, the ingestion of lipids in male Wistar rats was decreased by 40% below controls, while the other dietary components were ingested at exactly the same level as in animals fed ad libitum. After 7 weeks of treatment the liver mitochondria of lipid‐restricted animals showed significant increases in oxygen consumption with complex I‐linked substrates (pyruvate/malate and glutamate/malate). Neither mitochondrial H2O2 production nor oxidative damage to mitochondrial or nuclear DNA was modified in lipid‐restricted animals. Oxidative damage to mitochondrial DNA was one order of magnitude higher than that of nuclear DNA in both dietary groups. These results deny a role for lipids and reinforce the possible role of dietary proteins as being responsible for the decrease in mitochondrial ROS production and DNA damage in caloric restriction.

Effect of 8.5% and 25% caloric restriction on mitochondrial free radical production and oxidative stress in rat liver

Previous studies have consistently shown that 40% caloric restriction (CR) decreases the rate of mitochondrial ROS production and steady-state levels of markers of oxidative damage to macromolecules including mitochondrial DNA. However, few investigations have studied whether these changes also occur in lower CR regimes. This is of potential interest since moderate levels of dietary restriction are more practicable for humans. In this investigation male Wistar rats were subjected to 8.5% and 25% caloric restriction. Neither 8.5% nor 25% CR changed mitochondrial ROS production, oxygen consumption or mtDNA oxidative damage in rat liver mitochondria. However, both 8.5% and 25% CR significantly decreased the five different markers of protein oxidation, glycoxidation and lipoxidation measured, aminoadipic and glutamic semialdehyde, carboxyethyl-lysine, carboxymethyl-lysine, and malondialdehyde-lysine. The fatty acid composition of liver mitochondria was also affected and led to a moderate decrease in the degree of membrane unsaturation in both 8.5% and 25% CR. While 8.5% CR only affected complex I concentration (which was decreased), 25% CR decreased complexes I and IV and increased complexes II and III of the respiratory chain. Apoptosis-inducing factor (AIF) significantly decreased in 25% CR but not in 8.5% CR. The results show that moderate levels of caloric restriction can have beneficial effects including decreases in oxidative protein modification and a lower sensitivity of membranes to lipid peroxidation, in association with a reprogramming of the respiratory chain complexes and AIF content.

Mitochondrial DNA sequences are present inside nuclear DNA in rat tissues and increase with age.

Mitochondrial DNA (mtDNA) mutations increase with age. However, the number of cells with predominantly mutated mtDNA is small in old animals. Here a new hypothesis is proposed: mtDNA fragments may insert into nuclear DNA contributing to aging and related diseases by alterations in the nucleus. Real-time PCR quantification shows that sequences of cytochrome oxidase III and 16S rRNA from mtDNA are present in highly purified nuclei from liver and brain in young and old rats. The sequences of these insertions revealed that they contain single nucleotide polymorphisms identical to those present in mtDNA of the same animal. Interestingly, the amount of mitochondrial sequences in nuclear DNA increases with age in both tissues. In situ hybridization of mtDNA to nuclear DNA confirms the presence of mtDNA sequences inside nuclear DNA in rat hepatocytes. Bone marrow metaphase cells from both young and old rats show mtDNA at centromeric regions in 20 out of the 2n=40 chromosomes. Consequently, mitochondria can be a major trigger of aging but the final target could also be the nucleus.

La restricción de metionina en la dieta disminuye el estrés oxidativo en mitocondrias de corazón

Resumen Introduccion la restriccion calorica (RC) disminuye la produccion mitocondrial de radicales libres (MitROS) y el dano oxidativo al ADN mitocondrial (ADNmt) y aumenta la longevidad maxima, pero no se conocen los mecanismos implicados. Hemos encontrado que la restriccion de proteinas (RP) tambien produce esos cambios. Varios hallazgos relacionan a la metionina con el envejecimiento, y la restriccion de metionina (RMet) tambien aumenta, como la RC y la RP, la longevidad maxima. Hemos hipotetizado, por tanto, que la RMet es la causa del descenso en produccion de MitROS y estres oxidativo que ocurre en la RP y la RC. Material y metodos ratas Wistar macho sometidas a RMet o alimentadas ad libitum durante 7 semanas. Se aislaron mitocondrias funcionales de corazon por centrifugacion. La respiracion mitocondrial se midio por polarografia con electrodo de Clark y la produccion de MitROS por fluorometria. El dano oxidativo al ADNmt (8-oxodG) se midio por HPLC con deteccion electroquimica. Resultados la RMet disminuyo la produccion mitocondrial de ROS en el complejo I, no cambio el consumo de oxigeno mitocondrial y disminuyo los valores de la 8-oxodG en el ADNmt. Conclusiones los cambios observados en la produccion de MitROS y la 8-oxodG durante la RMet son iguales a los observados previamente en la RC y la RP. Esto sugiere que el descenso en la ingestion de metionina es la causa del descenso en la generacion de ROS y estres oxidativo mitocondrial y de parte del descenso de velocidad del envejecimiento que ocurre durante la restriccion calorica. La restriccion proteica (o de metionina) ofrece por primera vez una intervencion aplicable a las poblaciones occidentales, incluida la espanola, que es potencialmente capaz de retrasar en aproximadamente un 20% la velocidad del proceso endogeno del envejecimiento y de todas las enfermedades degenerativas asociadas a el sin necesidad de disminuir en nada la ingesta calorica.