Re: Amino acids - methionine restriction

[Guest guest]

The possible implications of the study that Al posted are very big.

" 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 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 " .

If proven, this would change the practice of CRON. We would be fine

tuning our calorie restriction by making sure to also restrict

methionine (and perhaps protein).

Thanks to Al and Rodney for bringing this issue to our attention.

If you are purposely restricting your protein intake, or your

methionine intake (apart from general calorie restriction), it would

be nice if you would please some details of exactly what you are

doing

thanks

Bob B.

>

> Hi All,

>  

> 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, López- M, Naudí 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±6.5 g in the

CONTROL and 245.0±6.2 g in the RESTAAS group at the beginning of the

experiment (non-significant difference), and 397.3±8.4 g and

381.3±7.2 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 0.864

> L-Histidine 0.33 0.198

> L-Leucine 1.11 0.666

> L-Isoleucine 0.82 0.492

> L-Valine 0.82 0.492

> L-Threonine 0.82 0.492

> L-Tryptophan 0.18 0.108

> L-Methionine 0.86 0.86

> L- Phenylalanine 1.16 0.696

> L-Arginine 1.12 0.672

> L-Glycine 2.33 1.398

> L-Cystine 0.34 0.204

> L-Proline 0.34 0.204

> L-Tyrosine 0.34 0.204

> L-Glutamic acid 2.70 1.620

> L-Aspartic acid 0.34 0.204

> L-Alanine 0.34 0.204

> L-Serine 0.34 0.204

> L-Asparagine 0.60 0.360

> Dextrine 5 5

> Corn starch 40.97 47.158

> Sucrose 20 20

> Corn oil 8 8

> Cellulose 5 5

> Choline bitartrate 0.2 0.2

> Vitamin mix (AIN 93G) 1 1

> Mineral mix (AIN 93G) 3.5 3.5

> Total (% 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 × 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±10.6 184.8±9.8

> SAH 39.8±4.6 34.4±4.0

> Ratio SAM/SAH 5.63±0.47 5.65±0.43

> ==============================================

>    Results are expressed in nmol/g tissue. Values are means±SEM

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±0.89 1.30±0.46*

> ADP 10.73±1.21 7.15±0.79*

> AMP 2.51±0.18 3.05±0.27

> Total adenine nucleotides 17.16±2.18 11.50±1.16*

> Ratio ATP/ADP 0.34±0.04 0.16±0.04**

> Adenylate energy charge 0.53±0.02 0.41±0.03**

> ==============================================

>    Values are means±SEM 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 ×

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±0.01 0.02±0.01

> Glu/mal 0.08±0.01 0.07±0.01

> Succinate 0.23±0.02 0.19±0.02

> Succinate + rotenone 0.09±0.02 0.08±0.02

> Pyr/mal + rotenone 0.14±0.01 0.12±0.01

> Succinate + Antimycin A 0.83±0.04 0.84±0.06

> ==============================================

>    Values are means±SEM 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±0.50 5.50±0.60

> Complex I-30 kDa subunit (NDUFS3) 100±8.77 87.38±4.17

> Complex I-39 kDa subunit (NDUFA9) 100±9.57 94.45±3.08

> Complex II-70 kDa subunit (Flavoprotein) 100±11.51 125.67±8.13

> Complex III-29 kDa subunit (Rieske iron-sulfur protein) 100±11.44

85.19±5.55

> Complex III-48 kDa subunit (CORE II) 100±8.64 80.09±4.30

> Complex IV-57 kDa subunit (COX I) 100±5.94 90.10±13.80

> PGC-1alpha 100±12.45 102.22±7.73

> NRF-2 100±21.67 100.96±13.16

> ==============================================

>    Values are means±SEM 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±0.04 0.31±0.05

> 16:0 16.63±0.27 16.84±0.39

> 16:1n-7 1.29±0.13 1.34±0.12

> 18:0 18.68±0.48 18.19±0.24

> 18:1n-9 7.97±0.30 8.50±0.19

> 18:2n-6 15.11±0.13 16.38±0.41*

> 18:4n-6 0.89±0.07 1.04±0.08

> 20:0 0.20±0.05 0.15±0.02

> 20:1 0.26±0.03 0.22±0.006

> 20:2n-6 0.61±0.07 0.59±0.02

> 20:3n-6 0.26±0.04 0.35±0.02

> 20:4n-6 30.31±0.22 28.98±0.31**

> 22:0 1.08±0.05 1.28±0.08

> 24:0 0.42±0.07 0.49±0.03

> 22:6n-3 5.93±0.26 5.27±0.18

> ACL 18.57±0.01 18.52±0.01*

> SFA 37.31±0.26 37.28±0.36

> UFA 62.68±0.26 62.71±0.36

> MUFA 9.53±0.43 10.07±0.27

> PUFA 53.14±0.28 52.64±0.45

> PUFAn-6 47.20±0.29 47.36±0.56

> PUFAn-3 5.93±0.26 5.27±0.18

> DBI 202.25±1.20 196.89±0.81**

> PI 188.83±1.91 180.29±0.65**

> Ratio 16:1/16:0 0.07±0.007 0.07±0.007

> Ratio 18:1/18:0 0.43±0.02 0.46±0.01

> Ratio 20:4/18:2 2.00±0.02 1.77±0.04**

> Ratio 20:4/20:3 132.11±20.63 83.87±6.90

> =======================================

>    Values are means±SEM 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@...

>

[Guest guest]

methionine restriction. I know you have disciseed this before but I was curious as to the limit you place on methionine in terms of grams per day? Positive Dennis