Amino acids - methionine restriction

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Results in the pdf-availed below paper using all except methionine amino acids restricted feeding without CR seem to find some, especially mitochondrial protein oxidation, apoptosis inducing factor and SIRT1, levels.

Effect of 40% restriction of dietary amino acids (except methionine) on mitochondrial oxidative stress and biogenesis, AIF and SIRT1 in rat liver.Caro P, Gomez J, I, R, Lˆupez- M, Naudˆq A, Portero-Otin M, Pamplona R, Barja G.Biogerontology. 2008 Nov 28. [Epub ahead of print]PMID: 19039676

Abstract

Previous studies have shown that the decrease in mitochondrial reactive oxygen species (mitROS) generation and oxidative damage to mitochondrial DNA (mtDNA) that occurs during life extending dietary restriction also occurs during protein or methionine restriction, whereas it does not take place during carbohydrate or lipid restriction.

In order to study the possible effects of other amino acids, in this investigation all the dietary amino acids, except methionine, were restricted by 40% in male Wistar rats (RESTAAS group). After 6-7 weeks, experimental parameters were measured in the liver.

Amino acid restriction did not change the levels of the methionine metabolites S-adenosylmethionine and S-adenosylhomocysteine, mitochondrial oxygen consumption and ROS generation, oxidative damage to mtDNA, amounts of the respiratory complexes I-IV, and the mitochondrial biogenesis factors PGC-1alpha and NRF-2. On the other hand, adenylate energy charge, mitochondrial protein oxidation, lipooxidation and glycooxidation, the degree of mitochondrial fatty acid unsaturation, and the amount of the apoptosis inducing factor (AIF) were decreased in the RESTAAS group. Amino acid restriction also increased SIRT1 protein.

These results, together with previous ones, strongly suggest that the decrease in mitROS generation and oxidative damage to mtDNA that occurs during dietary restriction is due to restriction of a single amino acid: methionine. They also show for the first time that restriction of dietary amino acids different from methionine decreases mitochondrial protein oxidative modification and AIF, and increases SIRT1, in rat liver.

Keywords: Free radical generation, Protein restriction, Aging, Protein damage, Fatty acid unsaturation, Mitochondrial respiratory complexes.

Abbreviations: ACL, acyl chain length, AIF Apoptosis inducing factor, AASA Aminoadipic semialdehyde in proteins, CEL Carboxyethyl-lysine in proteins, CML Carboxymethyl-lysine in proteins, DBI Double bond index, mtDNA Mitochondrial DNA, NRF-2 nuclear respiratory factor-2, GSA Glutamic semialdehyde in proteins, MDAL Malondialdehyde-lysine in proteins, PGC-1alpha PPAR-gamma Coactivator 1alpha, PI Peroxidizability index, ROS Reactive oxygen species, SAM S-Adenosylmethionine, SAH S-Adenosylhomocysteine, SIRT1 Sirtuin 1, 8-oxodG 8-Oxo-7,8-dihydro-2¡¬ deoxyguanosine.

Introduction

Aging increases the probability of suffering degenerative chronic diseases and death. It is important to clarify the main molecular mechanisms responsible for aging. Different kinds of evidence suggest that free radicals of mitochondrial origin are related to the aging process (Beckman and Ames 1998; Barja 2004a; Muller et al. 2007). Previous comparative studies consistently indicate that long-lived animals including mammals have low rates of reactive oxygen species (ROS) generation in the mitochondria of the main vital tissues (Barja 2004b) together with lower levels of oxidative damage in their mitochondrial DNA (mtDNA) measured as 8-oxo-7,8-dihydro-2¡¬ deoxyguanosine (8-oxodG). These two main characteristics can lead to a slower rate of accumulation of mitochondrial DNA mutations during life and thus to a longer life span (Barja 2004a). In addition, long-lived animals also have less unsaturated fatty acids in their mitochondria and other

cellular membranes, which protects them against lipid peroxidation and the negative impact of its products on other macromolecules including proteins and likely DNA (Pamplona et al. 2002a; Hulbert et al. 2007). It is interesting that the best-known experimental manipulation that extends life span, dietary restriction, also lowers mitochondrial ROS (mitROS) generation and 8-oxodG in mtDNA (Gredilla and Barja 2005). Dietary restriction also decreases oxidative, lipoxidative and glycoxidative damage to tissue and mitochondrial proteins (Pamplona et al. 2002b).

Recent studies from our laboratory have shown that protein (Sanz et al. 2004) or methionine (Sanz et al. 2006a) restriction also decrease mitochondrial ROS production and 8-oxodG in rat tissues, whereas these decreases do not occur during lipid (Sanz et al. 2006b) or carbohydrate (Sanz et al. 2006c) restriction. In addition, there is evidence indicating that dietary, protein, or methionine restriction, but not carbohydrate or lipid restriction, increase maximum longevity in mammals. A recent literature update showed that 16 out of 18 long-life survival experiments of protein restriction in rats or mice resulted in increases in maximum longevity of a mean intensity around half of that typically found during dietary restriction (Pamplona and Barja 2006). And longevity experiments have shown that methionine restriction also increases rat (Richie et al. 1994) and mouse ( et al. 2005) maximum longevity. These results, taken together, raise the

possibility that among the many different food components, the lower dietary intake of methionine can be the single factor responsible for the decrease in mitROS generation and oxidative DNA damage, and for part of the increase in longevity, during dietary restriction. But before this conclusion can be reached it must be investigated if other amino acid components of dietary protein also have an influence in those parameters, which has never been studied.

Therefore, in this investigation male Wistar rats were treated with semipurified diets in which all the amino acids except methionine were restricted by 40% (RESTAAS group) compared to the control diet. The study lasted 6-7 weeks and was performed in the liver since it is known that 40% protein (Sanz et al. 2004) or 40% methionine (Sanz et al. 2006a; Caro et al. 2008) restriction during this time period lowers mitROS generation and 8-oxodG in mtDNA in rat liver, similarly to what happens after 24 months of dietary restriction (- et al. 2002). Thus, 6-7 weeks are enough to detect the decrease in mitochondrial ROS generation and oxidative stress, and were then selected for the present study. At the end of the experimental period, in order to verify the constancy of methionine metabolism in the two experimental groups, two main methionine cycle metabolites, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were measured, and

adenine nucleotides were also analyzed in order to check for bioenergetic effects of the dietary treatment at tissue level. Mitochondrial oxygen consumption, mitROS production, oxidative damage to mtDNA and specific markers of protein oxidation, lipoxidation and glycoxidation were also measured. Since protein lipoxidation can be secondarily influenced by sensitivity to lipid peroxidation, the full fatty acid composition of mitochondrial membranes, and their degree of unsaturation was also measured. Since previous studies have shown that changes in mitROS generation can be due in part to variations in the amounts of respiratory complexes, the levels of complexes I to IV were also studied, as well as the factors controlling mitochondrial biogenesis PPAR-gamma coactivator 1alpha (PGC-1alpha) and nuclear respiratory factor-2 (NRF-2), which have been found to be elevated in dietary (Nisoli et al. 2005) or methionine (Naudi et al. 2007) restriction in

rodents. On the other hand, dietary restriction brings about many beneficial changes which can be due to changes in different dietary components in each case. The present experiment was then also used to clarify if the changes in apoptosis inducing factor (AIF) or sirtuin 1 (SIRT1) proteins are related or not to restriction of dietary amino acids different from methionine, which is presently unknown.

Methods

Animals and diets

Male Wistar rats of 6-7 weeks of age were caged individually ... After one week of acclimatization they were divided in two groups, CONTROL and RESTAAS (restricted in all the dietary amino acids except methionine). Semipurified diets prepared by MP Biochemicals (Irvine, CA, USA) were used. The mean body weight was 256.1¡Ó6.5 g in the CONTROL and 245.0¡Ó6.2 g in the RESTAAS group at the beginning of the experiment (non-significant difference), and 397.3¡Ó8.4 g and 381.3¡Ó7.2 g respectively at the end of the experimental period. Total body weight and food intake did not significantly differ between CONTROL and RESTAAS animals at any of the seven weeks of experimentation (results not shown) The diets contained a mixture of amino acids instead of protein. The detailed composition of the two diets is shown in Table 1. The RESTAAS diet contained 40% less of all the amino acids except methionine that was present at exactly the same level in the two diets.

The total decrease in amino acid content in the RESTAAS diet was compensated by increasing the amount of corn starch from 40.97 to 47.158%. Previous studies have shown that 40% variation in dietary carbohydrate does not modify mitochondrial H2O2 generation and 8-oxodG in the mtDNA in the liver of male Wistar rats (Sanz et al. 2006c). All the rest of the dietary components, corn oil, sucrose, dextrine, cellulose, choline bitartrate and vitamin and mineral mixes were maintained at exactly the same level in both diets. The CONTROL group received each day the same amount of food that the RESTAAS animals had eaten as a mean the previous week (pair feeding). After 6-7 weeks of dietary treatment the animals, which had 13-15 weeks of age, were ...

Table 1. Detailed composition of the semipurified diets used in this study: CONTROL, or 40% restricted in all the dietary amino acids except methionine (RESTAAS).==============================================Dietary component CONTROL (g/100 g) RESTAAS (g/100 g)==============================================L-Lysine 1.44 0.864L-Histidine 0.33 0.198L-Leucine 1.11 0.666L-Isoleucine 0.82 0.492L-Valine 0.82 0.492L-Threonine 0.82 0.492L-Tryptophan 0.18 0.108L-Methionine 0.86 0.86L- Phenylalanine 1.16 0.696L-Arginine 1.12 0.672L-Glycine 2.33 1.398L-Cystine 0.34 0.204L-Proline 0.34 0.204L-Tyrosine 0.34 0.204L-Glutamic acid 2.70 1.620L-Aspartic acid 0.34 0.204L-Alanine 0.34 0.204L-Serine 0.34 0.204L-Asparagine 0.60 0.360Dextrine 5 5Corn starch 40.97 47.158Sucrose 20 20Corn oil 8 8Cellulose 5 5Choline bitartrate 0.2 0.2Vitamin mix (AIN 93G) 1

1Mineral mix (AIN 93G) 3.5 3.5Total (% weight) 100 100

....

Results

The treatment of 40% restriction of all the dietary amino acids except methionine (RESTAAS group) did not cause significant changes in the methionine cycle metabolites SAM, SAH or their ratio (Table 2). The levels of liver total adenine nucleotides, ATP and ADP, but not of AMP, were significantly decreased in the RESTAAS group compared to controls (Table 3). In addition, the RESTAAS group showed a 53% decrease in ATP/ADP ratio and a 23% decrease in adenylate energy charge [(ATP + 0.5 ¡Ñ ADP)/(ATP + ADP + AMP)] compared to controls.

Table 2. Liver methionine metabolites S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) and their ratio in CONTROL and 40% amino acid restricted (RESTAAS) rats.============================================== CONTROL RESTAAS ==============================================SAM 201.8¡Ó10.6 184.8¡Ó9.8 SAH 39.8¡Ó4.6 34.4¡Ó4.0 Ratio SAM/SAH 5.63¡Ó0.47 5.65¡Ó0.43============================================== Results are expressed in nmol/g tissue. Values are means¡ÓSEM from 7-8 different animals. The rats in the RESTAAS group were dietary restricted by 40% in all the amino acids except methionine. No significant differences between groups were observed

Table 3. Liver adenine nucleotide contents and energy charge in CONTROL and 40% amino acid restricted (RESTAAS) rats.============================================== CONTROL RESTAAS==============================================ATP 3.91¡Ó0.89 1.30¡Ó0.46* ADP 10.73¡Ó1.21 7.15¡Ó0.79* AMP 2.51¡Ó0.18 3.05¡Ó0.27 Total adenine nucleotides 17.16¡Ó2.18 11.50¡Ó1.16* Ratio ATP/ADP 0.34¡Ó0.04 0.16¡Ó0.04** Adenylate energy charge 0.53¡Ó0.02 0.41¡Ó0.03**============================================== Values are means¡ÓSEM from 7-8 different animals. The rats in the RESTAAS group were dietary restricted by 40% in all the amino acids except methionine. ATP, ADP, AMP and Total adeninde nucleotide levels are expressed in nanomoles/mg protein. Total adenine nucleotides: ATP + ADP + AMP; Adenylate energy charge: (ATP + 0.5 ¡Ñ ADP)/(ATP + ADP + AMP).

Asterisks represent significant differences between the CONTROL and the RESTAAS groups: * (P <0.05), ** (P <0.01).

The rates of basal and stimulated mitochondrial ROS production with different substrate and inhibitor combinations are shown in Table 4. No significant differences in basal H2O2 production were observed between dietary groups with either complex I- or complex II-linked substrates (pyruvate/malate, glutamate/malate, succinate + rotenone, or succinate). The stimulated rates of H2O2 generation (pyruvate/malate + rotenone and succinate + antimycin A) did not show significant differences between groups either (Table 4). The rates of mitochondrial oxygen consumption did not show differences between dietary groups with any substrate either in state 4 (substrate alone) or in the phosphorylating state 3 (results not shown).

Table 4. Rates of H2O2 production (nanomoles H2O2/min mg protein) with different substrates or respiratory chain inhibitors in liver mitochondria from CONTROL and 40% amino acid restricted (RESTAAS) rats.============================================== CONTROL RESTAAS==============================================Pyr/mal 0.03¡Ó0.01 0.02¡Ó0.01 Glu/mal 0.08¡Ó0.01 0.07¡Ó0.01 Succinate 0.23¡Ó0.02 0.19¡Ó0.02 Succinate + rotenone 0.09¡Ó0.02 0.08¡Ó0.02 Pyr/mal + rotenone 0.14¡Ó0.01 0.12¡Ó0.01 Succinate + Antimycin A 0.83¡Ó0.04 0.84¡Ó0.06 ============================================== Values are means¡ÓSEM from 8 different animals. Pyr/mal Pyruvate/malate; Glu/mal Glutamate/malate. The rats in the RESTAAS group were dietary restricted by 40% in all the amino acids except methionine. No significant differences between groups were found with any

substrate or inhibitor combination

Oxidative damage to mitochondrial DNA (8-oxodG) did not show significant differences between dietary groups, and the same was observed for the amounts of the four complexes of the respiratory chain as well as for the mitochondrial biogenesis factors PGC-1alpha and NRF-2 (Table 5). On the other hand, AIF significantly decreased (by 18%) and SIRT1 significantly increased (by 26%) in the RESTAAS group compared to the controls (Fig. 1). ... * P <0.05.

Table 5. Oxidative damage to mtDNA (8-oxodG), mitochondrial respiratory complexes and biogenesis factors in liver of CONTROL and 40% amino acid restricted (RESTAAS) rats.============================================== CONTROL RESTAAS==============================================8-oxodG 5.97¡Ó0.50 5.50¡Ó0.60 Complex I-30 kDa subunit (NDUFS3) 100¡Ó8.77 87.38¡Ó4.17 Complex I-39 kDa subunit (NDUFA9) 100¡Ó9.57 94.45¡Ó3.08 Complex II-70 kDa subunit (Flavoprotein) 100¡Ó11.51 125.67¡Ó8.13 Complex III-29 kDa subunit (Rieske iron-sulfur protein) 100¡Ó11.44 85.19¡Ó5.55 Complex III-48 kDa subunit (CORE II) 100¡Ó8.64 80.09¡Ó4.30 Complex IV-57 kDa subunit (COX I) 100¡Ó5.94 90.10¡Ó13.80 PGC-1alpha 100¡Ó12.45 102.22¡Ó7.73 NRF-2 100¡Ó21.67 100.96¡Ó13.16 ============================================== Values are means¡ÓSEM from 5-8 different

animals. The rats in the RESTAAS group were dietary restricted by 40% in all the amino acids except methionine. Except in the case of 8-oxodG the units are: densitometry ratio of complex I, II, III, and IV/porin, and densitometry ratio PGC-1alpha and NRF-2/actin, and are expressed in RESTAAS animals in reference to CONTROLS (100%). 8-oxodG vales are expressed as 8-oxodG/10^5dG

The five different specific markers of protein oxidation, glycoxidation and lipoxidation are shown in Fig. 2. GSA, AASA, CEL, CML and MDAL showed significantly lower values in the RESTAAS than in the CONTROL group. These decreases ranged from a 21% decrease in CML to a 39% decrease in AASA, with a mean decrease of 28% for the five protein markers taken together. ... * P <0.05, ** P <0.01, *** P <0.001. ...

The full fatty acid composition of liver mitochondrial lipids is shown in Table 6. Among the different fatty acids, significant changes were observed only for linoleic acid (18:2n-6, which increased in the RESTAAS group) and for arachidonic acid (20:4n-6, which decreased in the RESTAAS group). This resulted in significant decreases in the total number of double bonds (DBI) and in the peroxidizability index (PI) in the RESTAAS group, while the total amount of saturated (SFA), unsaturated (UFA) or polyunsaturated (PUFA) fatty acids did not change. The ratio 20:4/18:2, which is indicative of delta-6 desaturase activity, significantly decreased in the RESTAAS group. The mean acyl chain length (ACL) showed a marginal decrease (of 0.003%) in RESTAAS animals, probably due to the partial substitution of 20:4n-6 for 18:2n-6 in the RESTAAS group.

Table 6. Fatty acyl composition (mol %) of total lipids in liver mitochondria from CONTROL and 40% amino acid restricted (RESTAAS) rats.======================================= CONTROL RESTAAS=======================================14:0 0.27¡Ó0.04 0.31¡Ó0.0516:0 16.63¡Ó0.27 16.84¡Ó0.3916:1n-7 1.29¡Ó0.13 1.34¡Ó0.1218:0 18.68¡Ó0.48 18.19¡Ó0.2418:1n-9 7.97¡Ó0.30 8.50¡Ó0.1918:2n-6 15.11¡Ó0.13 16.38¡Ó0.41*18:4n-6 0.89¡Ó0.07 1.04¡Ó0.0820:0 0.20¡Ó0.05 0.15¡Ó0.0220:1 0.26¡Ó0.03 0.22¡Ó0.00620:2n-6 0.61¡Ó0.07 0.59¡Ó0.0220:3n-6 0.26¡Ó0.04 0.35¡Ó0.0220:4n-6 30.31¡Ó0.22 28.98¡Ó0.31**22:0 1.08¡Ó0.05 1.28¡Ó0.0824:0 0.42¡Ó0.07 0.49¡Ó0.0322:6n-3 5.93¡Ó0.26 5.27¡Ó0.18ACL 18.57¡Ó0.01 18.52¡Ó0.01*SFA 37.31¡Ó0.26 37.28¡Ó0.36UFA 62.68¡Ó0.26 62.71¡Ó0.36MUFA 9.53¡Ó0.43 10.07¡Ó0.27PUFA 53.14¡Ó0.28 52.64¡Ó0.45PUFAn-6 47.20¡Ó0.29

47.36¡Ó0.56PUFAn-3 5.93¡Ó0.26 5.27¡Ó0.18DBI 202.25¡Ó1.20 196.89¡Ó0.81**PI 188.83¡Ó1.91 180.29¡Ó0.65**Ratio 16:1/16:0 0.07¡Ó0.007 0.07¡Ó0.007Ratio 18:1/18:0 0.43¡Ó0.02 0.46¡Ó0.01Ratio 20:4/18:2 2.00¡Ó0.02 1.77¡Ó0.04**Ratio 20:4/20:3 132.11¡Ó20.63 83.87¡Ó6.90======================================= Values are means¡ÓSEM from n = 7-8 different animals. The rats in the RESTAAS group were dietary restricted by 40% in all the amino acids except methionine. ... Asterisks represent significant differences between the control and the RESTAAS groups: * P <0.05, ** P <0.01

Discussion

In this investigation, restriction of dietary amino acids different from methionine is performed for the first time in relation to mitochondrial oxidative stress, apoptosis and biogenesis, concerning the role of dietary restriction in longevity extension. The treatment brought about consequences at tissue level as evidenced by the changes in adenine nucleotides. The animals in the RESTAAS group had lower levels of total adenine nucleotides (Table 3). This was due to a decrease in ADP and ATP, not in AMP. In addition, the ATP/ADP ratio was lower in RESTAAS animals and the same was true for the adenylate energy charge. Therefore, the treatment caused a decrease in the steady-state content of biological energy in the liver tissue. This seems to be due to the restriction of essential amino acids other than methionine. It is known that limitation of essential amino acids initiates an amino acid response pathway that responds to amino acid deficiency by

regulating many steps along the pathway of DNA to RNA to protein (Kilberg et al. 2005). These regulated events include chromatin remodeling, changes in mRNA and translation leading to variations in expression of many proteins including metabolic enzymes. On the other hand, it is well known that changes in dietary methionine lead to changes in methionine cycle metabolites including SAM and SAH (Finkelstein and 1986). In this investigation, no differences between dietary groups in either liver SAM or SAH, or in SAM/SAH ratio were observed (Table 2), in agreement with the presence of methionine at the same concentration in both diets. Therefore, both the adenylate and the SAM and SAH results indicate that the dietary model was effective at tissue level.

We show here that restriction of dietary amino acids except methionine does not change the rate of mitROS generation. Many different studies from different laboratories have consistently shown that dietary restriction lowers mitROS generation in many tissues including liver (reviewed in Gredilla and Barja 2005). Furthermore, it has been observed that protein restriction also lowers mitROS production (Sanz et al. 2004) whereas this does not occur after restricting only the lipids (Sanz et al. 2006b) or the carbohydrates (Sanz et al. 2006c) in the diet. Thus, it is the protein component of the diet that is responsible for this phenomenon. Further studies showed that restricting only dietary methionine also lowered mitROS generation (Sanz et al. 2006a; Caro et al. 2008). In agreement with the changes observed for mitROS production, there is evidence indicating that dietary, protein, or methionine restriction, but not carbohydrate or lipid restriction,

increase maximum longevity in laboratory rodents (reviewed in Pamplona and Barja 2006). The decrease in mitROS generation during methionine restriction suggested that this single dietary amino acid could be responsible for the decrease in ROS production both during dietary and protein restriction. But before this conclusion could be reached it was necessary to study the possible effect of the other dietary amino acids. In the present investigation, the lack of effect of restriction of all the dietary amino acids different from methionine rules out such possibility and finally demonstrates, for the first time, that the reduced ingestion of methionine is solely responsible for the decrease in the rate of mitochondrial free radical generation.

In agreement with the lack of effect on mitROS generation, the level of 8-oxodG in mtDNA was not different in the RESTAAS than in the CONTROL group. Previous studies have found a close correlation between 8-oxodG in mtDNA and mitROS production in restriction models. Thus, similarly to mitROS generation, oxidative damage in mtDNA decreases in dietary (- et al. 2002), protein (Sanz et al. 2004) and methionine (Sanz et al. 2006a; Caro et al. 2008) restriction, whereas it does not change in carbohydrate (Sanz et al. 2006c) or lipid (Sanz et al. 2006b) restriction or in RESTAAS animals (this investigation). Taken together, those results indicate that the reduced ingestion of methionine during dietary restriction is the single dietary factor responsible for the decrease in mitROS generation and oxidative damage to mtDNA, which can lower the rate of accumulation of mtDNA mutations and thus contribute to life span extension (Barja 2004a).

Concerning mechanisms responsible for the decrease in mitROS generation during methionine restriction, previous studies have shown that it is due in part to variations in the levels of respiratory chain complexes (Caro et al. 2008), particularly to changes in the amounts of complexes I (mainly) and III, since these two complexes are the ones responsible for ROS generation in the respiratory chain. In the present investigation, in agreement with the lack of variations in mitROS generation in the RESTAAS group, there were no changes in the amounts of complex I or III. On the other hand, in further agreement with the lack of changes in any of the four complexes of the respiratory chain, no changes in either PGC-1alpha or in NRF-2 were observed in the RESTAAS group. Respiratory chain gene expression is under the control of the PGC-1 family of coactivators including PGC-1alpha, which responds to environmental stimuli like cold or food availability, and

in turn target specific transcription factors like NRF-2 controlling the expression of genes coding for the mitochondrial respiratory chain ( and Hoogenraad 2007). Previous studies have found an increase in mitochondrial biogenesis during dietary restriction (Nisoli et al. 2005), but it is not know what dietary components are responsible for this response. The results of the present investigation rule out the role of amino acids different from methionine, whereas in a single study in rat brain we found increases in the amount of PGC-1alpha during methionine restriction (Naudi et al. 2007). Therefore, dietary methionine could be responsible at least in part for the increase in mitochondrial biogenesis during dietary restriction, although clearly much additional work is needed before this conclusion can be reached.

This investigation was also useful to clarify if known variations in other parameters relevant for longevity extension during dietary restriction are due to changes in dietary amino acids different form methionine. Previous investigations have shown that protein oxidation, glycoxidation and lipoxidation, measured as GSA, AASA, CEL, CML, and MDAL in mitochondrial proteins are lowered by dietary (Pamplona et al 2002b), protein (Ayala et al. 2007), and methionine (Sanz et al. 2006a) restriction in the rat, thus contributing to improve the oxidative status in the restricted animals. In the present investigation all these protein damage markers were decreased after restriction of dietary amino acids different from methionine (Fig. 2). Therefore, not only methionine restriction, but also the restriction of other dietary amino acids is able to induce protective effects on mitochondrial proteins. Since in the RESTAAS group the decreases in protein

modification occurred without variation in mitROS generation, this is probably due to an increase in degradation of modified proteins instead of a lowered rate of formation of protein oxidative modifications. On the other hand, some of the protein markers measured (CML and MDAL) are dependent on lipid peroxidation. Since it is well known that lipid peroxidation increases strongly as a function of the number of double bonds per fatty acid, we measured the full fatty acid composition of liver mitochondria (Table 6). We found that the RESTAAS treatment significantly decreased the total number of double bonds (DBI) as well as the peroxidizability index (PI). Thus, part of the decrease in CML and MDAL can be secondary to this decrease in fatty acid unsaturation. But this can not be the full explanation because the other three protein markers measured, which do not depend on lipid peroxidation (GSA, AASA and CEL), were also decreased in the RESTAAS group. In

any case and independently from the molecular mechanism involved, the generalized decrease in protein oxidative modification induced by restriction of dietary amino acids different from methionine can contribute to the decrease in aging rate that takes place during dietary restriction.

The decrease in fatty acid unsaturation in RESTAAS mitochondria was due to variations in only two fatty acids which are linked in the same metabolic biosynthetic route. RESTAAS animals showed a decrease in the highly unsaturated 20:4n-6 and an increase in its less unsaturated precursor 18:2n-6, without changes in the total amount of saturated or unsaturated fatty acids. This kind of change has been also observed when comparing the fatty acid composition of long-lived with that of short-lived mammalian species (Pamplona et al. 2002a; Hulbert et al. 2007). Their lower degree of unsaturation confers to the membranes of long-lived animals a superior resistance to lipid peroxidation and lower lipoxidation-dependent damage to macromolecules. In the present investigation the change from 20:4n-6 to 18:2n-6 induced by RESTAAS seems to be due to a decrease in delta-6 desaturase activity, a main limiting enzyme in the n-6 biosynthesis pathway, since 20:4/18:2

was the only fatty acid ratio that was significantly decreased by amino acid restriction. The decrease in fatty acid unsaturation in the RESTAAS group can, in concert with the decrease in mitochondrial ROS generation, increase longevity during dietary restriction (which includes amino acid restriction). Although the change in DBI was small (around 3%), its effect on lipid peroxidation can be much larger since it is known that the increase in lipid peroxidation rate as the number of fatty acid double bonds increases is not linear but tends to be rather exponential (Holman 1954).

Concerning AIF, recent studies suggest that this mitochondrial protein has both life and death functions in cells (Porter and Urbano 2006). A certain amount of AIF is required for oxidative phosphorylation, and AIF-deficient cells exhibit a reduced content of complex I (Vahsen et al. 2004), pointing to a role for AIF in the biogenesis of this complex. In our study AIF was decreased by 18% in RESTAAS animals (Fig. 1), but the amount of complex I did not vary. This indicates that such level of decrease in AIF is not large enough to become limitant for complex I biogenesis. On the other hand, since AIF also has proapototic functions, our results suggests that restriction of amino acids different from methionine can contribute to increased protection from tissue apoptosis through moderate decreases in AIF that do not compromise respiratory chain function.

Silent information regulator 2 (sir 2) genes in yeast, Drosophila and C. elegans encode NAD + -dependent histone deacetylases and have roles in lifespan determination and response to dietary restriction (Rogina and Helfand 2004; Chen and Guarente 2007). In mammals Sirtuins 1-7 are homologues of Sir 2 proteins, SIRT1 is the family member with highest homology to Sir 2 and is considered its orthologue (Frye 2000). SIRT1 can also deacetylate histones and other proteins (Vaquero et al. 2004). Recent studies suggest that SIRT1, like sir2 in yeast and invertebrates, is involved in life extension induced by dietary restriction in mammals, an experimental manipulation that increases SIRT1 levels (Boily et al. 2008). However, it has never been studied what dietary components are responsible for increases in sirtuin proteins during dietary restriction. In the present investigation the RESTAAS animals showed a significant increase (26%) in SIRT1 in rat liver.

This shows that it is not necessary to decrease caloric ingestion to induce SIRT1, and indicates that restriction of dietary amino acids different from methionine has the capacity to elicit such change. This does not eliminate the possibility that other dietary components are also additionally involved in elevations of SIRT1 during dietary restriction, an aspect that should be studied in future investigations.

Finally, recent studies indicate that SIRT1 directly interacts with and deacetylates PGC-1alpha (Nemoto et al. 2005) analogously to its interaction with p53 or members of the Forkhead transcription factor family like Foxo3a, and SIRT1-dependent cycles of PGC-1alpha acetylation and deacetylation seem to occur in the mammalian liver during the fed and fasted states (Rodgers et al. 2005). Therefore, the increase in SIRT1 could contribute to increase PGC-1alpha activity and thus transcription of mitochondrial genes and mitochondrial biogenesis during dietary restriction. However, no changes in PGC-1alpha or in the amounts of mitochondrial respiratory complexes were found in the RESTAAS group in the present investigation (Table 5) in spite of the increase in SIRT1. Various regulatory phenomena at molecular level can be responsible for this apparent discrepancy. On the one hand, the SIRT1-dependent PGC-1alpha deacetylation model is perhaps simplistic and

does not take into account other PGC-1alpha modifications such as phosphorylation and methylation (Rodgers et al. 2008). In addition, while activation of SIRT1 deacetylates PGC-1alpha, it also lowers its transcriptional activity and decreases cellular oxygen consumption by 25% (Nemoto et al. 2005). Various changes could occur at different extents during dietary restriction, a manipulation that increases PGC-1alpha and mitochondrial biogenesis, when compared with restricting only the dietary amino acids different from methionine. Moreover, in the particular case of liver, the increases in glucagon and glucocorticoid hormones during fasting or dietary restriction increase PGC-1alpha gene transcription (Rodgers et al. 2008), whereas these hormonal changes probably do not take place in our model of amino acid restriction.

In summary the results of this study, together with previous investigations, lead us to conclude that the decrease in mitROS generation and oxidative damage to mtDNA that seems to be implicated in life extension during dietary restriction in mammals is due to the reduced ingestion of a single dietary substance: methionine. Restriction of other dietary amino acids did not change mitROS production, 8-oxodG in mtDNA, mitochondrial oxygen consumption, amounts of the four complexes of the respiratory chain and mitochondrial biogenesis indicators. On the other hand, restriction of amino acids different to methionine can contribute to decreased levels of oxidatively modified proteins, to lower lipid peroxidation in vivo, decreased apoptosis inhibiting factor, and increased SIRT1. Therefore, at least due to these actions, it can collaborate to extend mammalian life span during dietary and protein restriction. In contrast, the decrease in adenylate energy

charge induced by amino acid restriction is not expected to contribute in that direction and could even limit the increase in life span observed during dietary or protein restriction.

-- Al Pater, alpater@...