Low methionine ingestion extends lifespan.

Abstract

Dietary energy restriction has been a widely used means of experimentally extending mammalian life span. We report here that lifelong reduction in the concentration of a single dietary component, the essential amino acid L-methionine, from 0.86 to 0.17% of the diet results in a 30% longer life span of male Fischer 344 rats. Methionine restriction completely abolished growth, although food intake was actually greater on a body weight basis. Studies of energy consumption in early life indicated that the energy intake of 0.17% methionine-fed animals was near normal for animals of their size, although consumption per animal was below that of the much larger 0.86% methionine-fed rats. Increasing the energy intake of rats fed 0.17% methionine failed to increase their rate of growth, whereas restricting 0.85% methionine-fed rats to the food intake of 0.17% methionine-fed animals did not materially reduce growth, indicating that food restriction was not a factor in life span extension in these experiments. The biochemically well-defined pathways of methionine metabolism and utilization offer the potential for uncovering the precise mechanism(s) underlying this specific dietary restriction-related extension of life span.

Full text

Nutrient Requirements and Interactions
Low Methionine Ingestion by Rats Extends Life Span
NORMAN ORENTREICH,1 JONATHAN R. MATÕAS, ANTHONY DEFELICE AND
JAY A. ZIMMERMAN
Orentreich Foundation for the Advancement of Science, Inc., BiomédicalResearch Station,
Cold Spring-on-Hudson, NY 10516
ABSTRACT Dietary energy restriction has been a
widely used means of experimentally extending mam
malian life span. We report here that lifelong reduction in
the concentration of a single dietary component, the
essential amino acid L-methionine, from 0.86 to 0.17%
of the diet results in a 30% longer life span of male
Fischer 344 rats. Methionine restriction completely abol
ished growth, although food intake was actually greater
on a body weight basis. Studies of energy consumption
in early life indicated that the energy intake of 0.17%
methionine-fed animals was near normal for animals of
their size, although consumption per animal was below
that of the much larger 0.86% methionine-fed rats. In
creasing the energy intake of rats fed 0.17% methionine
failed to increase their rate of growth, whereas res
tricting 0.85% methionine-fed rats to the food intake of
0.17% methionine-fed animals did not materially reduce
growth, indicating that food restriction was not a factor
in life span extension in these experiments. The bio
chemically well-defined pathways of methionine metab
olism and utilization offer the potential for uncovering
the precise mechanism(s) underlying this specific di
etary restriction-related extension of life span. J. Nutr.
123: 269-274, 1993.
INDEXINGKEY WORDS:
•dietary restriction •life span extension
•aging •methionine •rats
As first shown by McCay more than 50 years ago
(McCay 1935) and by many others since, the most
effective and most widely used experimental means
of extending life span is by restriction of energy
intake (Masoro 1988, Weindruch 1990). A wide va
riety of species have been studied, and in nearly every
case a reduction in energy intake has been associated
with an extension of life span. Although there is little
debate about the beneficial effects of such restriction,
which include such varied effects as delayed immune
senescence (Eberly and Bruckner Kardoss 1989),
retardation of cancer development (Cohen et al. 1988,
Klurfeld et al. 1989), alterations in gene expression
(Semsei et al. 1989), improved antioxidant protection
(Laganiere and Yu 1989) and enhanced DNA repair
(Srivastava and Busbee 1992), there remains con
siderable uncertainty about the mechanism! s)through
which these varied effects are attained.
Early studies of energy restriction indicated that to
be maximally effective in extending life span, re
striction needed to be initiated early enough and se
verely enough to retard growth (Beauchene et al.
1986, McCay 1935). Nevertheless, beneficial effects
have been reported when restriction was first imposed
in adult rats (Weindruch and Walford 1982). The
reduction in growth seen in many experiments in
which restriction was initiated early in life is
probably a marker of an alteration in some fun
damental developmental and/or gerontologie process,
and is seen as reduced growth only so long as such
growth is possible. Apparently, once growth potential
is exhausted, it is still possible to prolong life, but
retarded growth no longer serves as an indicator.
In view of the growth-retarding nature of energy
restriction in young animals, we have examined other
nontoxic strategies for reducing growth in order to
determine whether such approaches could also extend
life span. We report here that feeding purified isoca-
loric diets deficient in the essential amino acid
methionine eliminates growth and markedly im
proves survival of rats.
METHODS
These studies were reviewed and approved by the
Institutional Animal Care and Use Committee of the
Orentreich Foundation for the Advancement of
Science, Inc.
To study survival of rats fed low methionine for
extended periods of time, 60 Fischer 344 male rats
obtained from Taconic Farms (Germantown, NY) at 4
'To whom correspondence should be addressed.
0022-3166/93 $3.00 ©1993 American Institute of Nutrition. Received 6 August 1992. Accepted 14 October 1992.
269
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270 ORENTREICH ET AL.
wk of age and prefed for 2 wk a nonpurified diet
(Ralston Purina, St. Louis, MO) were randomly as
signed to one of two groups receiving a purified diet
containing either 0.86% or 0.17% L-methionine
(Table 1). Groups of five animals were housed in a
conventional animal facility in solid-bottomed cages
lined with wood chips. Temperature was maintained
at 22°C, and lighting was on for 12 h/d. Unless
otherwise specified, all animals were given free access
to food and acidified water throughout the study.
When food consumption was measured, the food
ration for each cage of five rats was weighed at the
initiation of the feeding interval, and again 48 h later,
at which time the animals also were weighed. Food
intake was measured twice per week during the first 2
mo of the experiment, and other cohorts were
measured at later times of life.
Because pilot studies had indicated that rats fed
0.17% methionine consume less food than do animals
fed 0.85% methionine in the diet, we examined the
effects of energy intake per se in two types of experi
ments in which either 0.17% methionine-fed rats
consumed an energy-dense diet to compensate for
their reduced food intake, or 0.86% methionine-fed
TABLE 1
Composition of control diet1
IngredientL-ArginineL-LysineL-HistidineL-LeucineL-IsoleucineL-ValineL-Methionine2L-ThreonineL-TryptophanL-PhenylalanineGlycineGlutamic
acid2DextrinCornstarchSucroseSolka-FlocCholine
bitartrateVitamin
mix3Mineral
mix4Corn
oilAmountg/kg11.214.43.311.18.28.28.68.21.811.623.327.050.0436.1200.050.02.010.035.080.0
Manufactured by Ziegler Brothers (Gardners, PA) as extruded
pellets, except when the energy content was raised by the addition
of corn oil, in which case the diets (control and elevated energy
density) were prepared as powdered meal.
2When the methionine content of the diet was reduced, the
glutamic acid content was raised on an equal gram basis.
^AIN-76â„¢ vitamin mix (AIN 1977) except that the concen
tration of menaquinone was 50 mg/kg.
4AIN-76â„¢ mineral mix (AIN 1977).
-o- 0.86% MET
-A- o.17% MET
100
80
'> 60
W 40
20
O
50
300 550 800
Age (days)
1050
1300
FIGURE 1 Survival of Fischer 344 male rats fed 0.86% or
0.17% methionine beginning at 42 d of age (n - 30 rats in
each group).
rodents were limited to the amount of food consumed
by animals fed the low methionine ration.
In studies in which energy-dense diets were em
ployed, 10 animals were fed for 3 mo a 0.17% L-
methionine diet identical to that used throughout
these studies, with the exception that the corn oil
concentration of the diet was increased (at the ex
pense of Solka-Floc) such that the energy density was
raised from the normal level of 17.9 to 19.7 kj/g. In
0.17% methionine-fed rats this level offsets the
reduction in energy intake relative to those animals
fed 0.86% methionine in the diet during the first 90 d
of feeding.
To limit 0.86% methionine-fed rats to the intake
of animals receiving 0.17% methionine, food intake
was measured in 10 singly housed young rats
receiving 0.17% methionine, and their average food
intake was then offered to 10 singly housed rats
receiving the 0.86% methionine ration. A third group
of individually housed rats received free access to
0.17% methionine-containing diet. Initially, when
the animals were growing rapidly, food intake was
measured every 24 h; later, when growth had slowed,
food intake was measured weekly, but feeding of the
paired animals was always on a daily basis.
Statistical methods. Analysis of survival was con
ducted using Gehan's Wilcoxon test (Lee 1980), as
implemented in True Epistat (Epistat Services,
Richardson, TX). All other comparisons were per
formed using the Student's t test. Differences be
tween groups were considered to be significant when
P < 0.01.
RESULTS
Effect of dietary methionine on survival. Rats fed
low methionine (0.17%) starting at 4-6 wk of age
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LIFE SPAN EXTENSION AND METHIONINE
271
-°- 0.86% MET
-A- Q.17% MET
500
~ 400
a
O)
•
>>
-a
o
m
300
200
100
,-A-A-A-A-A-A-A-A-A-A-A-A
140
2BO 420
560 700
Age (days)
FIGURE 2 Growth of Fischer 344 male rats fed 0.86 or
0.17% methionine beginning at 42 d of age. Values are
means ±SEM, n = 30 rats in each group. In some instances
error bars are not visible.
showed greater median (1059 vs. 818 d) and
maximum (1252 vs. 1116 d) life spans than those fed
0.86% methionine (Fig. 1).When the methionine con
centration of the diet was reduced below 0.12% no
rats survived for longer than 1 mo (data not shown).
Effect of dietary methionine on growth. Rats fed
0.86% methionine from 42 d of age gained nearly 350
g during the next 50 wk of the experiment. On the
other hand, rats fed a 0.17% methionine diet from 42
d of age failed to gain weight throughout their lives
(Fig. 2).
At the end of 90 d of feeding 0.17% methionine to
rats, the reproductive organs (testes, seminal vesicles)
were smaller and lung and heart were larger (relative
to body size) than in rats fed 0.86% methionine. The
relative sizes of the liver, prostate gland and spleen
were unchanged by methionine restriction (Table 2).
Food intake. Food intake was measured twice
weekly for the first 2 mo of feeding, again at the end
of 3 mo of feeding and, in another cohort of animals,
at 24 mo of age. Rats fed 0.17% methionine con
sumed 10% less food than control animals during the
first 2 mo, 12% less food at 3 mo and 24.5% less food
at 24 mo of age. Expressed in terms of body weight,
however, after 3 mo of feeding, 0.17%
methionine-fed animals had eaten 93% more food per
gram of body weight (8.3 ±0.4 vs. 4.3 ±0.2 g
food-d-MOO g body wtr1) than rats given 0.86%
methionine (Fig. 3). By 24 mo of age this difference
was reduced to a 62% greater intake per gram of body
weight in the 0.17% methionine-fed rats (data not
shown).
To assess the importance of energy intake per se on
growth in the 0.17% methionine-fed rats, 10 animals
were fed a 0.17% methionine diet identical to that
used throughout these studies, with the exception
that the energy density was raised from 17.9 to 19.7
-°- 0.86% MET -*- 0.17% MET
400
» 300
*•
£
S
« 200
>»
oc-0'
,5-5'
555-522-522-2
15 30 45 00 75 90
Days of Feeding
20
(8
S
JD 10
0
•e
o
o
15 30 45 6(
Days of Feeding
75 90
is
12
o
5 9
s
O
•a
O
o
u.
15 30 46
60 75
90
Days of Feeding
FIGURE 3 Growth and food intake of 6-wk-old male
Fischer 344 rats fed 0.86% methionine for 15 d, then either
0.86 or 0.17% methionine. Top: Body weight. Middle: Food
intake per animal. Bottom: Food intake per 100 g of body
weight (BW). Values are means ±SEM, n = 10. In some
instances error bars are not visible.
kj/g. This level compensates for the lower energy
intake of 0.17% methionine-fed rats at 3 mo of age.
Animals consuming this energy-dense 0.17%
methionine diet consumed the same amount of ration
as did those fed the diet containing 17.9 kj/g, but they
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272
ORENTREICH ET AL.
TABLE 2
Relative organ size in Fischer 344 rats consuming diets containing 0.86 or 0.17% methionine for 90 </'
Diet (energy content]
Body weight,gOrgan
weight, g/100 g bodywtTestesProstateSeminal
vesiclesLiverSpleenLungHeartFat
pad:RightLeft0.86%
Met
(17.9kj/g)258
±6.0*1.61
±0.04*0.06
±0.010.29
±0.06*3.56
±0.130.21
±0.010.42
±0.02*0.28
±0.01*1.26
±0.09*1.37
±0.11*0.17%
Met
117.9kj/g)118
±6.01.03
±0.020.05
±0.010.10
±0.023.25
±0.110.23
±0.010.53
±0.010.38
±0.120.58
±0.120.61
±0.110.17%
Met
(19.7kj/g)123
±8.31.16
±0.020.05
±0.010.10
±0.013.18
±0.130.22
±0.010.50
±0.020.50
±0.220.51
±0.100.61
±0.13
Values are means ±SEM,n = 10; "P < 0.01 vs. the other two groups.
failed to gain weight during 3 mo of feeding (data not
shown). Further, relative organ sizes in rats fed the
energy-dense 0.17% methionine diet were not signifi
cantly different from those of animals fed the non-
energy-dense 0.17% methionine diet (Table 2).
To further evaluate the role of food intake in
methionine restriction, we limited 0.86%
methionine-fed rats to the reduced amount of food
consumed by 0.17% methionine-fed animals. Fol
lowing a lag in growth, the pair-fed animals grew
rapidly, and by the end of the second month of
feeding they had attained the same body weight as the
cohort offered free access to 0.86% methionine-con-
taining ration,- body size in these two groups was
indistinguishable thereafter (Fig. 4).
DISCUSSION
For the past 50 years restricted energy intake has
been the principal effective method for experimen
tally extending life span. We report here that re
striction of a single dietary component, the essential
amino acid methionine, also prolongs life. This obser
vation may offer a new and valuable tool in ex
perimental gerontology because the precise mechan-
ism(s) underlying life span extension following energy
restriction is unknown and has proven difficult to
identify due to the relatively broad and ill-defined
roles of energy in biological systems. On the other
hand, the better-known metabolic pathways of
methionine metabolism(s) offer the possibility of de
termining the mechanism by which this particular
deprivation improves life expectancy.
In studies of the relationship between energy
intake and longevity, the restricted animals are
usually fed some fraction of the food consumed by
unrestricted control animals. Because the restricted
animals grow only to the extent that they are offered
nutrient, they grow less and, consequently, over a
lifetime consume less energy per animal, although the
energy intake per gram of body weight is not altered.
In sharp contrast, the animals in our studies were fed
a palatable diet in unrestricted quantities; they
cannot be called restricted in the conventional sense.
Indeed, although the food intake per animal was
modestly lower in those fed a 0.17% methionine diet,
food intake per gram of body weight was markedly
—O— 0.86% MET -A- 0.17% MET -A- Pair Fed
400
40 60
Days of Feeding
BO
100
FIGURE 4 Growth of Fischer 344 male rats given free
access to diet containing 0.86 or 0.17% methionine, or pair-
fed 0.86% methionine-containing diet in amounts limited
to those consumed by animals offered free access to diet
containing 0.17% methionine. Values are means ±SEM, n -
10 rats in each group. In some instances error bars are not
visible.
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LIFE SPAN EXTENSION AND METHIONINE
273
increased. Comparison of the food intake of large
animals with that of smaller animals, as is conven
tionally done in many life extension studies, is thus
flawed with respect to our studies because larger
animals will obviously consume more. Rather, the
determination of "restriction" should be in terms of
food intake by animals of the same size. On this basis,
the 0.17% methionine-fed rats in our studies con
sumed the same amount (or more) of energy as did
rats of the same size receiving a normal level (0.86%)
of methionine in their rations.
In an effort to further evaluate the role of energy
intake on methionine-related life span extension, we
limited 0.86% methionine-fed rats to the food intake
consumed by 0.17% methionine-fed animals. The
unimpaired growth of these rats argues that the
degree of food intake reduction (per animal) in our
long-term 0.17% methionine-fed animals is insuffi
cient to account for the life span extension observed
because, regardless of the restriction used (energy,
protein or methionine), life span is extended only at
nutrient intake levels that impair growth (in animals
with growth potential).
In attempting to elucidate factors responsible for
the life span-extending actions of dietary restriction,
dietary components other than energy have been
studied in the hope of identifying a single responsible
nutrient or class of nutrients. However, neither a
reduction in fat nor a reduction in mineral content
significantly altered survival in rats when energy
intake was held constant (Iwasaki et al. 1988a and
1988b). On the other hand, reduced protein intake has
been associated with modest life span extension (Leto
et al. 1976, Masoro et al. 1991), possibly attributable
to delayed nephrotoxicity (Masoro et al. 1991).
However, the magnitude of life span extension seen
in rats fed 0.17% methionine in our studies was
considerably greater than that attained with protein
restriction. Further, with the exception of the
methionine and glutamic acid (which replaces
methionine in the 0.17% methionine diet) content,
the amino acid composition of both the experimental
and control diets was identical, and the nitrogen
content was unchanged, eliminating any putative ef
fects of the nitrogen content of the diets. We therefore
do not believe that delayed nephrotoxicity explains
the life span prolongation observed in rats fed a 0.17%
methionine diet.
Previous reports have indicated that a diet deficient
in tryptophan extends life span in rats (Ooka et al.
1988, Segall and Timiras 1976, Timiras et al. 1984).In
view of our observation of life span extension when
methionine is reduced in the diet, there is then a
suggestion that deprivation of single essential amino
acids at a level consistent with survival (but not with
growth) is capable of producing life span extension.
We do not yet know whether this might be a general
feature of essential amino acid restriction or whether
methionine acts through some mechanism unique to
itself.
Thus, at this time we cannot identify the exact
mechanism(s) underlying the improved survival seen
in rats fed reduced levels of methionine. Indeed, many
of the predicted actions of prolonged sulfhydryl amino
acid deficiency would shorten life. That the mech
anism of the life extension is not yet known should
not deter the use of the methionine-restricted rat
model in aging research, because energy restriction,
the only other paradigm in wide use for modifying
aging, is also unexplained. Further, animals raised on
the restricted methionine protocol can be housed in
groups and are given free access to food, minimizing
housing and husbandry costs associated with such
long-term studies.
It is entirely possible that energy and methionine
restriction approaches to life span enhancement act
through some common final pathway. Should that be
the case, comparison of the physiologic and bio
chemical effects of these two models might reveal
those pathways that are essential to enhance life
span, while distinguishing them from those that are
of minor importance. Further, by focusing on specific
biochemical pathways it may be possible to identify
more precisely the specific mechanism) s) responsible
for life span extension under conditions of energy
deprivation and/or decreased methionine intake.
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