It's Never Too Late: Calorie Restriction is Effective in Older Mammals

Abstract

Calorie restriction (CR – the selective reduction of energy intake without compromising other essential nutrients), is the most powerful intervention known to retard biological aging in mammals. Yet although CR research dates back nearly 70 years, it was regarded largely as a laboratory curiosity for most of that time, because of the belief that CR worked through retarding growth early in the life history, and therefore had no relevance for intervention in aging in adults. A landmark report by Weindruch and Walford overturned this belief unambiguously demonstrating that with a suitably-modified protocol, CR would retard aging and extend lifespan in young-adult mice. This report launched the modern era of CR research. There have remained, however, reasons to doubt that CR would work in substantially older organisms. A new report from Steven Spindler’s group answers the question, showing that it is possible to achieve decisive extensions of mean and maximal lifespan and significant early reductions in cancer-associated mortality, even in animals in early seniority. This article places this new report in the context of previous research and discusses its implications for biomedical intervention in the degenerative aging process.

Full text

REJUVENATION RESEARCH
Volume 7, Number 1, 2004
© Mary Ann Liebert, Inc.
Perspective
It’s Never Too Late: Calorie Restriction is
Effective in Older Mammals
MICHAEL RAE
3
C
ALORIE RESTRICTION
(CR), the selective re-
duction of energy intake without compro-
mising other essential nutrients, is the most
powerful intervention known to retard biolog-
ical aging in mammals, as assessed by exten-
sion of mean and maximum lifespan, reduced
incidence or progression of age-associated dis-
eases, and preserved physiological function
and molecular fidelity with age.
1
Yet although
CR research dates back nearly 70 years, it was
regarded largely as a laboratory curiosity for
most of that time, because of the belief that CR
worked through retarding growth early in the
life history. This notion was reinforced by nu-
merous failed attempts to replicate CR’s anti-
aging effects when the regimen was instituted
in adult organisms (reviewed in Ref. 2).
Twenty years ago, this belief was overturned
by a classic report by Weindruch and Walford,
2
who were the first to unambiguously demon-
strate that, if imposed gradually and with a
generous provision of essential nutrients (so as
to allow for the lesser metabolic adaptability of
older organisms), adult-onset CR could exert
the same robust anti-aging effects observed
when the regimen is implemented in wean-
lings. The result initiated a new era of interest
in CR as a method of experimental manipula-
tion of the aging process—both as tool for in-
vestigating the mechanisms of aging, and in
hopes of designing alternative interventions
which might exploit the mechanisms of CR
to extend healthy lifespan in humans (“CR
mimetics”
3
).
There have remained, however, reasons for
caution regarding the efficacy of CR in older
organisms. Reasonable grounds for scepticism
included limits on older animals’ ability to
metabolically adapt to the CR regime, given
young adult animals’ evidently reduced ca-
pacity in this regard relative to weanlings; the
ability of late-life CR to meaningfully improve
functionality, given the previous accumulation
of a lifetime of molecular disarray; the time re-
quired, relative to the remaining life ex-
pectancy of animals in late middle age, for CR’s
anti-aging mechanisms to translate into clini-
cally significant functional improvements rela-
tive to an ad libitum (AL) cohort; and the es-
tablished fact of CR’s age-retarding benefits
being proportional to the time an organism
spends on a CR regimen (Fig. 1).
Studies on surrogate outcomes also sug-
gested limits on late-life CR’s effectiveness.
Thus CR was found to rapidly reduce levels
of carbonyl and loss of sulfhydryl groups in
the brain, but was unable to preserve cardiac
sulfhydryl groups;
4
CR lowered the level of al-
tered heat-labile hepatic, renal, and cerebral
proteins, and hepatic mitochondrial (but not
cytosolic) carbonylated proteins;
5
and while
one year of late-onset CR reduced “ragged red”
muscle fiber segments and mitochondrial DNA
deletions, this effect manifested at 50%, but not
The Calorie Restriction Society, Gardena, California.

at 35%, CR
6
– and 6 weeks’ CR feeding, when
initiated at 18–22 months, was unable to reduce
mitochondrial protein carbonyls or loss of
sulfhydryl groups.
7
The results overall dem-
onstrate a rapid reduction in oxidative stress,
but confirm the expectation that much pre-
existing, accumulating oxidative damage is left
unaffected by at least short- to medium-term
CR implemented in older animals. Simultane-
ously, the shift in steady-state REDOX tone
underlying these results would be expected
to reduce the secondary age-related shifts in
REDOX-sensitive gene transcription (briefly re-
viewed in Ref. 8) And indeed, Dr. Stephen
Spindler’s laboratory at UC Riverside recently
issued an important report of the predicted
rapid shift in gene expression in older mice
subjected to CR.
9
But the strongest reason to regard the search
for CR mimetics as a dubious endeavor has
been a series of reported failures of late-life CR
to clearly extend lifespan. Yet these studies
could not be considered to be definitive refu-
tations of late-life CR’s efficacy, because all of
them repeated methodological errors high-
lighted by Weindruch and Walford
2
with re-
spect to provision of nutrients and gradual im-
position of CR,
10,11
leaving the question of CR’s
effects in older organisms open.
A new report by Spindler’s group
12
is there-
fore of considerable significance. Using a care-
ful experimental design reflecting the insights
of Weindruch and Walford’s groundbreaking
experiment,
2
these investigators initiated CR at
19 months of age and achieved decisive exten-
sions of mean and maximal lifespan, relative to
both intraexperimental (extensions of
15%)
and historical
13
controls, accompanied by sig-
nificant early reductions in cancer-associated
mortality.
Additionally, Dhahbi et al.
12
performed a mi-
croarray analysis on hepatic gene expression of
late-onset CR animals after 2, 4, and 8 weeks of
intervention, and found that CR rapidly in-
duced shifts in gene expression away from the
AL profile which parallel 72% of the changes
observed in animals maintained on CR from
the age of 7 months onward.
In principle, this design should lead to in-
formation of considerable value. As the inves-
tigators note, most previous gene expression
studies have been cross-sectional comparisons
of expression profiles of young AL vs. old AL
and CR animals. This method produces data of
RAE
4
FIG. 1. Relationship between the duration of CR and maximum LS.
19

little ultimate value, as the results do not allow
one to distinguish expression shifts which are
causal in the anti-aging action of CR from those
which are its effects—a fact that has often been
glossed over in previous discussion of these
findings. Indeed, examining the gene chip pro-
file of an aged organism would reasonably be
predicted to primarily reveal compensatory
adaptations to the primary, accumulated mo-
lecular lesions that define the aging process,
and to confirm that CR animals have been less
subject to those lesions over their lifetimes.
By demonstrating a panel of gene expression
changes which are closely temporally linked to
extension of lifespan, the results of Dhahbi et
al.’s could, in principle, allow for both greater
confidence in assigning a causal role for those
changes in the resulting lifespan (LS) gains, and
for the use of this profile as a positive control
snapshot against which to test putative CR
mimetics (a task to which Spindler’s group has
already begun to apply these data).
12
However,
the design of the gene chip study necessitates
caution in accepting the data for this purpose.
One group of methodological concerns re-
lates to the age of the animals used in the two
arms of the studies. Whereas CR was initiated
at 19 months for the LS study [a time point
which, as the authors take care to point out,
was 2 months before the visible acceleration
of age-related mortality in the animals—the
“knee” in the survival curve], animals used for
gene expression studies were all sacrificed for
that purpose after 2, 4, or 8 weeks’ CR initiated
at age
32 mo, to create what is in fact a short
cross-sectional series rather than a truly longi-
tudinal investigation.
The considerably greater age of the animals
used for the gene expression study can be ex-
pected to distort the results. On the one hand,
the
basal gene expression profile to which the
post-CR profile is ultimately compared can
only be expected to differ in magnitude, and
perhaps even in its very existence, as in the case
of age-related hypo- or
de novo hyper-methyla-
tion.
14
If so, then the relative (“-fold”) changes
in expression observed upon implementation
of CR—and perhaps even the fact of those
changes—can only be predicted to be partially
artifacts of this aspect of the design.
On the other hand, the greater age of the an-
imals may also be expected to alter the re-
sponses of those organisms to CR. For instance,
considerably older animals may fail to make
some of the metabolic adaptations necessary
for the manifestation of retarded aging, or the
time course of that shift may be considerably
altered. The fact that CR fails when initiated in
older organisms unless special care is taken to
impose the regimen gradually and to ensure
the nutrient quality of diet appears to testify to
just such a reduction in the metabolic flexibil-
ity required for adaptation to the diet.
We cannot, in fact, even be confident that
animals this old are even
capable of making
such a shift and, therefore, that their gene ex-
pression profiles will accurately reflect those
changes essential to the process—precisely be-
cause we do not have lifespan data for such a
study.
A second, more minor methodological weak-
ness of the new study
12
relates to the decision
to have all animals at the full level of restric-
tion ultimately achieved in the LS study at the
time of sacrifice for microarray analysis.
11
This
protocol led to CR being implemented on a
time scale different from that actually used for
the lifespan study, which, in turn, may be pre-
dicted to distort the results obtained.
Following the example set by Weindruch and
Walford’s successful protocols,
2
the animals
used in the LS study had their caloric intake re-
duced in two steps: caloric intake was reduced
by 17% for two weeks, following which the full
44% CR regimen was imposed. By contrast, in
order to have animals fully on CR in time for
the gene chip studies, the animals sacrificed at
2 weeks underwent the first reduction in caloric
intake for one week, followed by a second week
at the fully restricted intake. This accelerated
initiation of CR may have altered the magni-
tude, time-course, or even the fact of some
genes’ differential expression under CR. This
seems particularly likely in the case of genes re-
ported as “oscillators.”
A possible counterargument to all of the
above objections would be that 72% of the dif-
ferences in gene expression observed in life-
long CR vs. AL animals were recapitulated in
the late-onset CR animals. But to take the op-
posing view, the fact that over a quarter of all
the gene changes observed following long-term
CR are not reproduced in the late-onset group
leaves open the possibility that at least some of
CALORIE RESTRICTION IN OLDER MAMMALS
5

T
ABLE
1. E
FFECT OF
C
ALORIE
R
ESTRICTION
(CR) I
NITIATED AT
V
ARIOUS
A
GES ON
L
IFESPAN
*
Age
Study initiated kcal/wk % AL
a
Mean Max
c
Mean Max
c
Mean Max
c
Mean Max
c
Weindruch et al.
20
28 days 50 59 43 51 30 28 45 29 31 28
(weaning)
Weindruch and Walford
2
12 mo 90 56 36.9 45.1 19 11 25 14 18 16
Pugh et al.
22
12 mo 62 74 32.6 41.8 13 10 11 10.6 16 16
Dhahbi et al.
12
19 mo 52.2 56 35.4 43.6 15 16 20 10 40 32
a
“Ad libitum” controls in all 3 studies were in fact restricted 10–25% from observed AL intake to avoid confound-
ing effects of obesity.
b
Computed by subtracting ages at death from age of initiation, as compared to the same subtraction in controls.
c
Maximum lifespans expressed as mean tenth-decile survivorship.
*All studies used similar, longevous hybrid genotypes: C3B10RF1
20,22
; B6C3 F1
12
; C57BL/6
22
.
NB: The known strain variability of the response to CR: Ref. 2 reported lower absolute lifespans, but greater rela-
tive extensions, in B6 mice than in C3B10RF1; cf. Ref. 21.
the gene expression changes essential to the
anti-aging effects of CR are among their num-
ber. (Valuable insight into these issues might
be gained by repeating the current study’s pro-
tocol in weanling mice, in whom LS-prolong-
ing CR is readily and consistently induced.)
An additional caveat is that the microarray
data collected was that of a particular organ,
the liver. While (as previous studies have
shown) many of the effects of CR on gene ex-
pression are broadly similar across tissues,
there are some clearly tissue-specific effects of
CR—and some of these may be critical to its ef-
fects on survivorship and physiology.
As one example of difficult-to-unravel issues
of tissue specificity and/or cause vs. effect, con-
sider the findings of Lee et al.,
15
who have
observed that genes associated with stress re-
sponse are upregulated with age in gastrocne-
mius muscle and in two regions of the brain
(neocortex and cerebellum),
16
but only the for-
mer demonstrated increases in expression of
inflammation-associated transcripts. In yet
greater contrast, long-term CR animals’ mus-
cles manifest increases in the expression of
genes involved in carbohydrate metabolism,
while the carbohydrate-metabolic class is
down-
regulated
in their brains. A snapshot of the
short-term effects of CR in these tissues might
lead to opposite inferences regarding a possi-
ble causal role of a CR-induced anti-inflamma-
tory response or alterations in carbohydrate
metabolism in its anti-aging action, and a pu-
tative CR mimetic which had no effect on lev-
els of expression of these inflammatory genes
would be judged either effective or not, de-
pending on which tissue was used as the ref-
erence standard.
These methodological issues call into ques-
tion the utility of the expression profile as a
screen for candidate CR mimetics.
The finding that CR can rapidly impact aging
and cancer, and video footage of the aged AL
and late-onset CR animals (which strongly pre-
sents a picture of more robust health in the CR
group)
17
combine to give reason for optimism
regarding the
efficacy of late-life CR, or of late-
life use of CR mimetics, which are heartening
findings for the growing body of human CR
practitioners,
18
and for biopharma labs and ven-
ture capitalists seeking to produce extremely
useful CR-mimetic compounds. It would be ex-
tremely useful in this context, to have a fuller
characterization of the effects of late-life CR on
molecular disorder and physiology.
There remains, however, the question of the
degree of clinical impact to be anticipated from
CR-based interventions when implemented in
persons in late middle age, such as the postwar
“baby boom” cohort whose entry into senior-
ity is in large part responsible for increasing in-
terest in truly interventional biogerontology.
It is widely held that the effectiveness of CR
lessens at later ages. While Dhahbi et al’s data
12
RAE
6
% Remaining
% AL
a
% Historical
13
LS
b
Lifespan
Energy intake (mo.)
Life extension

are compatible with this belief in an absolute
sense, results appear to demonstrate that the
effect on
remaining LS of late-onset CR similar
to that of CR initiated even shortly after wean-
ing (Table 1). In comparing the impact of CR
initiated at 12 months of age between refer-
ences 2 and 22, it is instructive to note that high
absolute “AL” (and hence CR) intake in Wein-
druch & Walford
2
relative to all other studies,
and the similar
absolute LS results from a lesser
reduction in
percentage intake from controls
(which yet resulted in a lower
absolute caloric
intake) in Ref. 22. Obviously interstudy com-
parisons are highly fraught, but it is worth not-
ing that the strain of mice used in the cited arm
of Ref. 2 is in fact normally more longevous
than that used in Ref. 22 (cf. Refs. 20 and 21).
The overall impact of the aggregate findings
seems to suggest a similar extension of re-
maining LS can be obtained at any time from
a similar absolute caloric intake. A separate pa-
per might be devoted to this surprising find,
which is counterintuitive to assumptions which
flow from a model of aging involving the ac-
cumulation of irreversible damage during ag-
ing, and which parallels less-surprising find-
ings recently reported in
Drosophila.
23
The
results of this comparison suggest, at mini-
mum, the optimistic conclusion that the poten-
tial effect of even young adult-onset CR has un-
til now been underestimated.
Dhahbi et al.,’s exciting results with late-life
CR,
12
while impressive and a reason for opti-
mism, must nonetheless remind us of the limi-
tations of a therapy which can only slow the pro-
gression of the disease which it treats (aging), as
opposed to one that can actually cure it.
11
Ulti-
mately, CR and CR mimetics are a limited anti-
aging intervention in whichever species they are
implemented. The emerging field of biomedical
gerontology must focus its attention on the de-
velopment of methods for the effective
removal
of existing, age-related molecular damage,
rather than to the
retardation of the mechanisms
that lead to or allow for its accrual. An appar-
ently exhaustive panel of such interventions has
been proposed;
24,25
and if it is such, then it
would almost tautologically constitute a means
to reverse and abolish biological aging, divorc-
ing the passage of time from the increasing risk
of disability and death.
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The Calorie Restriction Society
1827 W. 145
th
Street
Suite 205
Gardena, CA 90249
E-mail:
michaelrae@cadvision.com
RAE
8