Alberto Sanz

Caloric restriction decreases mitochondrial free radical generation at complex I and lowers oxidative damage to mitochondrial DNA in the rat heart

The effect of caloric restriction (CR) (40%) on the rates of mitochondrial H2O2 production and oxygen consumption and oxidative damage to nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) was studied for short‐term (6‐wk) and long‐term (1‐year) periods in the heart of young and old rats. Short‐term CR did not change any of the parameters measured. However, long‐term CR significantly decreased the rate of mitochondrial H2O2 generation (by 45%) and significantly lowered oxidative damage to mtDNA (by 30%) without modifying damage to nDNA. The decrease in H2O2 production occurred exclusively at the complex I free radical generator of the respiratory chain. The mechanism allowing that decrease was not a simple decrease in mitochondrial oxygen consumption. Instead, the mitochondria of caloric‐restricted animals released fewer oxygen radicals per unit electron flow in the respiratory chain. This was due to a decrease in the degree of reduction of the complex I generator in caloric‐restricted mitochondria. The results are consistent with the concept that CR decreases the aging rate at least in part by decreasing the rate of mitochondrial oxygen radical generation and then the rate of attack on mtDNA.

Influence of aging and long-term caloric restriction on oxygen radical generation and oxidative DNA damage in rat liver mitochondria.

The effect of long-term caloric restriction and aging on the rates of mitochondrial H2O2 production and oxygen consumption as well as on oxidative damage to nuclear (nDNA) and mitochondrial DNA (mtDNA) was studied in rat liver tissue. Long-term caloric restriction significantly decreased H2O2 production of rat liver mitochondria (47% reduction) and significantly reduced oxidative damage to mtDNA (46% reduction) with no changes in nDNA. The decrease in ROS production was located at complex I because it only took place with complex I-linked substrates (pyruvate/malate) but not with complex II-linked substrates (succinate). The mechanism responsible for that decrease in ROS production was not a decrease in mitochondrial oxygen consumption because it did not change after long-term restriction. Instead, the caloric restricted mitochondria released less ROS per unit electron flow, due to a decrease in the reduction degree of the complex I generator. On the other hand, increased ROS production with aging in state 3 was observed in succinate-supplemented mitochondria because old control animals were unable to suppress H2O2 production during the energy transition from state 4 to state 3. The levels of 8-oxodG in mtDNA increased with age in old animals and this increase was abolished by caloric restriction. These results support the idea that caloric restriction reduces the aging rate at least in part by decreasing the rate of mitochondrial ROS production and so, the rate of oxidative attack to biological macromolecules like mtDNA.

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)

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction, Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

Background Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established. Methodology/Principal Findings We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψm). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. Conclusions/Significance These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

The Mitochondrial Free Radical Theory of Aging: A Critical View

The Mitochondrial Free Radical Theory of Aging (MFRTA) proposes that mitochondrial free radicals, produced as by-products during normal metabolism, cause oxidative damage. According to MFRTA, the accumulation of this oxidative damage is the main driving force in the aging process. Although widely accepted, this theory remains unproven, because the evidence supporting it is largely correlative. For example, long-lived animals produce fewer free radicals and have lower oxidative damage levels in their tissues. However, this does not prove that free radical generation determines life span. In fact, the longest-living rodent -Heterocephalus glaber- produces high levels of free radicals and has significant oxidative damage levels in proteins, lipids and DNA. At its most orthodox MFRTA proposes that these free radicals damage mitochondrial DNA (mtDNA) and in turn provoke mutations that alter mitochondrial function (e.g. ATP production). According to this, oxidative damage to mtDNA negatively correlates with maximum life span in mammals. However, in contrast to MFRTA predictions, high levels of oxidative damage in mtDNA do not decrease longevity in mice. Moreover, mice with alterations in polymerase gamma (the mitochondrial DNA polymerase) accumulate 500 times higher levels of point mutations in mtDNA without suffering from accelerated aging. Dietary restriction (DR) is the only non-genetic treatment that clearly increases mean and maximum life span. According to MFRTA caloric restricted animals produce fewer mitochondrial reactive oxygen species (mtROS). However, DR alters more than free radical production (e.g. it decreases insulin signalling) and therefore the increase in longevity cannot be exclusively attributed to a decrease in mtROS generation. Thus, moderate exercise produces similar changes in free radical production and oxidative damage without increasing maximum life span. In summary, available data concerning the role of free radicals in longevity control are contradictory, and do not prove MFRTA. In fact, the only way to test this theory is by specifically decreasing mitochondrial free radical production without altering other physiological parameters (e.g. insulin signalling). If MFRTA is true animals producing fewer mtROS must have the ability to live much longer than their experimental controls.

Expression of the Ciona intestinalis Alternative Oxidase (AOX) in Drosophila Complements Defects in Mitochondrial Oxidative Phosphorylation

Defects in mitochondrial OXPHOS are associated with diverse and mostly intractable human disorders. The single-subunit alternative oxidase (AOX) found in many eukaryotes, but not in arthropods or vertebrates, offers a potential bypass of the OXPHOS cytochrome chain under conditions of pathological OXPHOS inhibition. We have engineered Ciona intestinalis AOX for conditional expression in Drosophila melanogaster. Ubiquitous AOX expression produced no detrimental phenotype in wild-type flies. However, mitochondrial suspensions from AOX-expressing flies exhibited a significant cyanide-resistant substrate oxidation, and the flies were partially resistant to both cyanide and antimycin. AOX expression was able to complement the semilethality of partial knockdown of both cyclope (COXVIc) and the complex IV assembly factor Surf1. It also rescued the locomotor defect and excess mitochondrial ROS production of flies mutated in dj-1beta, a Drosophila homolog of the human Parkinsons disease gene DJ1. AOX appears to offer promise as a wide-spectrum therapeutic tool in OXPHOS disorders.

Expression of the yeast NADH dehydrogenase Ndi1 in Drosophila confers increased lifespan independently of dietary restriction

Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling.

Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan

Summary Increased production of reactive oxygen species (ROS) has long been considered a cause of aging. However, recent studies have implicated ROS as essential secondary messengers. Here we show that the site of ROS production significantly contributes to their apparent dual nature. We report that ROS increase with age as mitochondrial function deteriorates. However, we also demonstrate that increasing ROS production specifically through respiratory complex I reverse electron transport extends Drosophila lifespan. Reverse electron transport rescued pathogenesis induced by severe oxidative stress, highlighting the importance of the site of ROS production in signaling. Furthermore, preventing ubiquinone reduction, through knockdown of PINK1, shortens lifespan and accelerates aging; phenotypes that are rescued by increasing reverse electron transport. These results illustrate that the source of a ROS signal is vital in determining its effects on cellular physiology and establish that manipulation of ubiquinone redox state is a valid strategy to delay aging.

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O2 consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (Nepsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.

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.