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studied in rat liver without changing the amount eaten per
day of the other dietary components and it was found, like
in 40 % DR, that 40 % PR decreases liver mitROSp and
FRL specifically at complex I, lowers 8-oxodG in mtDNA
(95, Table 1), and decreases five specific markers of
protein oxidative, glycoxidative and lipoxidative
modification as well as the amount of complex I protein in
rat liver mitochondria and tissue (96). Strikingly, the
direction of change, the magnitude, mechanisms, and site
of action exerted by PR on mitROSp and 8-oxodG in
mtDNA are almost identical to those found in 40 % DR
(58). Taken together, those studies suggest that proteins
are the dietary components responsible for most or all the
decrease in mitROSp and oxidative damage to mtDNA
that takes place in DR, as well as for part of the increase in short-lived mammals (rats and mice) of similar body size
longevity induced by this dietary intervention.
It was logical to suspect that dietary methionine could
be involved in those PR and DR effects since it was
already known that MetR, independently of energy
restriction, increases rat (maximum) longevity (56) while
such effect had not been described for any of the other
dietary amino acids. This is why the effects of MetR on
mitROSp and oxidative stress were studied at my
laboratory (Table 1). The results showed that isocaloric
MetR (40 % and 80 %), applied to young rats during 7
weeks, lowers mitROSp (mainly at complex I), the FRL,
the complex I content, 8-oxodG in mtDNA (Table 1), and
specific markers of protein oxidative, glycoxidative and
lipoxidative modification in rat heart (at 40 % and 80
%MetR; 97, 98) or liver (at 40 % MetR; 99, 100)
mitochondria, similarly to what occurs after 7 weeks of 40
% MetR in rat kidney and brain mitochondria (101, 102).
In order to obtain these decreases it was enough to restrict
methionine by 40 % (Table 1). Those decreases in
mitochondrial ROSp (at complex I) and oxidative stress
have recently been reproduced in liver mitochondria of
pigs subjected to MetR (103). 80 % MetR leaded to similar
decreases in 8-oxodG than in 40 % MetR, while the
decrease in mitROSp from controls to 40 % MetR rats was
more pronounced than that occurring from 40 % MetR to
80 %MetR.
Most importantly, and consistently with an important
role of methionine in the DR beneficial effects, when all
the dietary amino acids -except methionine- were
restricted (also by 40 %) during 7 weeks, neither the rate
of mitROSp nor the level of 8-oxodG in mtDNA were
modified (104). In addition, it was found that 40 %MetR
also decreases mitROSp, FRL and 8-oxodG in mtDNA
and reverses aging-related increases in protein
modification when implemented during only 7 weeks in 24
months old rats (99). All those results, taken together,
indicate that the lowered ingestion of methionine during
MetR (and PR and DR) is responsible for all or most of the
decreases in mitROSp and oxidative stress observed during
these three longevity extending manipulations. Such
lowered methionine ingestion is most likely also
responsible for all (during PR and MetR) or part (during
DR) of the life-extension effects observed during these
60
Gustavo Barja de Quiroga
dietary manipulations. This extraordinary capacity of a
single dietary molecule to induce the decrease in mitROSp
is still present when the animals reach old age.
Various mechanisms can be responsible for the
decrease in mtROSp during MetR. A most simple one
would be based on a decrease in the content of the
complex I protein in MetR that would directly lead to a
decreased rate of mitROSp. This has been reported under
40 % MetR in the majority of tissues studied (Table 1),
and also during DR and PR, as well as in long-lived birds
(pigeons, canaries and parakeets) compared to the much
and weight-specific metabolic rate (105, 106). But, in
principle, this would not be the full explanation because
MetR also induces qualitative changes in mitochondria
because it also decreases FRL. Such decrease could be due
to a decrease in the mid point potential -and thus the
degree of electronic reduction- of the complex I ROS
generator, because the decrease in mitROSp during MetR
is observed, like in DR, with partial complex I electronic
reduction (with complex I-linked substrates alone) but not
with full reduction (complex I-linked substrates plus
rotenone). The result is that MetR mitochondria (from both
young and old animals) are more efficient in avoiding
mtROS generation that those of ad libitum fed rats. MetR
mitochondria leak less radicals per unit of electron flow in
the respiratory chain, similarly to what has been found in
especially long-lived compared to short-lived animal
species (birds), as well as in DR and PR rats compared to
ad libitum fed ones (55). These quantitative and qualitative
changes could be due to: i) direct interaction of
methionine, or more likely, of a more chemically reactive
methionine metabolite, with the matrix domain complex I
polypeptide/s involved in ROS generation; ii) changes in
cellular signaling molecules and the ensuing modification
of specific gene expression of mitochondrial proteins; iii)
decreases in the concentration of mitochondrial complex I
substrates like pyruvate which would decrease matrix
NADH and are known to occur at least during DR (see
section 5).
Concerning the possible direct interaction of
methionine or its metabolites with mitochondria without
the information passing through the nucleus (mechanism
"i"), it is known that direct in vitro addition of methionine
to isolated functional rat mitochondria increases their rates
of mitROSp (87). Therefore a rather direct and rapid effect
of methionine on complex I seems to occur. A methionine
metabolite could be responsible for this effect because in
methionine, differing from homocysteine or cysteine, the
potentially reactive sulfur is located inside the molecule
and is therefore not available for direct covalent chemical
reaction with protein thiols. Interestingly, the reaction of
methionine with hydroxyl radicals generates methionine
radical carbon-, nitrogen- and sulfur-centered radicals as
intermediates in the formation of the methanetiol product,
as detected by EPR spin trap techniques and GC-FID and
GC-MS techniques (107). These radicals or methanetiol
(CH3SH) itself could react with complex I or some of its
@Real Academia Nacional de Farmacia. Spain