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2009 37: 47 originally published online 15 December 2008Toxicol Pathol
Rozalyn M. Anderson, Dhanansayan Shanmuganayagam and Richard Weindruch
Caloric Restriction and Aging: Studies in Mice and Monkeys
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Caloric Restriction and Aging: Studies in Mice and Monkeys
ROZALYN M. ANDERSON,1,2DHANANSAYAN SHANMUGANAYAGAM,1,2AND RICHARD WEINDRUCH1,2,3
1Wisconsin National Primate Research Center, Madison, Wisconsin, USA
2GRECC VA Hospital, Madison, Wisconsin, USA
3Department of Medicine, SMPH, University of Wisconsin, Madison, Wisconsin, USA
It is widely accepted that caloric restriction (CR) without malnutrition delays the onset of aging and extends lifespan in diverse animal models
including yeast, worms, flies, and laboratory rodents. The mechanism underlying this phenomenon is still unknown. We have hypothesized that a
reprogramming of energy metabolism is a key event in the mechanism of CR (Anderson and Weindruch 2007). Data will be presented from studies
of mice on CR, the results of which lend support to this hypothesis. Effects of long-term CR (but not short-term CR) on gene expression in white adi-
pose tissue (WAT) are overt. In mice and monkeys, a chronic 30% reduction in energy intake yields a decrease in adiposity of approximately 70%.
In mouse epididymal WAT, long-term CR causes overt shifts in the gene expression profile: CR increases the expression of genes involved in energy
metabolism (Higami et al. 2004), and it down-regulates the expression of more than 50 pro-inflammatory genes (Higami et al. 2006). Whether aging
retardation occurs in primates on CR is unknown. We have been investigating this issue in rhesus monkeys subjected to CR since 1989 and will dis-
cuss the current status of this project. A new finding from this project is that CR reduces the rate of age-associated muscle loss (sarcopenia) in mon-
keys (Colman et al. 2008).
caloric restriction; aging; mammals; primate; metabolism; PGC-1α.
emerged that longevity could be manipulated (McCay et al.
1935). Reduction of caloric intake without malnutrition extended
average and maximum lifespan in male and female rats and
delayed the onset of age-associated pathologies, suggesting
that the prospect of increased longevity was no longer a myth.
Since then, scientific and public interest in CR as a means to
extend lifespan has continued to grow. In rodents, many of
the diseases and disorders associated with age—including
cancer, obesity, diabetes, autoimmune diseases, sarcopenia and
cardiovascular disease—are opposed by CR (Masoro 2002;
Weindruch and Walford 1988). The impact of CR on these
conditions has important implications for human health and has
prompted further studies that can be divided into two groups.
The first are “early-onset CR” studies (e.g., start CR on one-
month-old, weanling rodents), which represent the best avail-
able model to study the biology of dietary-induced decelerated
aging in mammals; the second is “adult-onset CR” studies
(e.g., start CR in middle age), which are most relevant for
potential human application.
The relationship between caloric intake and lifespan was
investigated in female mice from the long-lived C3B10F1
hybrid strain (Weindruch et al. 1986). With conventional main-
tenance, mice are group housed and fed ad libitum. For dietary
studies, mice are individually housed, and food intake is regu-
lated (Pugh et al. 1999). This study led to the discovery that
caloric intake is inversely proportional to lifespan and compared
mice on an ad libitum diet (approximately 115 kcal/mouse/
week) to mice on a variety of restricted diets (85, 50, and 40
kcal/mouse/week). Nutrient-enriched diets were fed to the two
most severely restricted groups. The reduction in calorie intake
was reflected in the body weights of the four groups of mice.
Caloric restriction (CR) is the only dietary intervention
known to extend lifespan and delay the onset of age-associated
phenotypes (Masoro 2002; Weindruch and Walford 1988). The
retardation of aging by a simple reduction in calorie intake no
doubt belies the complexity of the underlying mechanisms.
Although the phenomenon was first described over seven
decades ago, the mechanistic basis remains elusive. In this
paper, we will present a brief overview of CR and describe
some key early studies in rodents that reveal the potential of this
regimen as a means to understand aging. Next, we will describe
the studies in mice that led us to the hypothesis that reprogram-
ming of energy metabolism is critical to the underlying mecha-
nisms of CR and present peroxisome proliferator activated
receptor-gamma coactivator 1-alpha (PGC-1α) as a candidate
regulator in this process. Finally, we will describe the relevance
of CR studies to human health and longevity, including data
from our two-decade-long study in rhesus monkeys and some of
the findings from shorter-term human studies.
AGING RETARDATION BY CR
Throughout history, there has been considerable interest in
identifying factors that confer increased longevity, from the
elixir of life to the fountain of youth. It was not until about
seventy-five years ago, however, that any credible evidence
Address correspondence to: Rozalyn Anderson, Ph.D., GRECC D5214,
VA Hospital, 2500 Overlook Terrace, Madison WI 53705, USA; e-mail:
Abbreviations: CR, caloric restriction; WAT, white adipose tissue.
Toxicologic Pathology, 37: 47-51, 2009
Copyright © 2009 by Society of Toxicologic Pathology
ISSN: 0192-6233 print / 1533-1601 online
48ANDERSON ET AL.TOXICOLOGIC PATHOLOGY
In addition, restricted mice lived longer than their ad libitum
counterparts in a manner that was inversely proportional to
their degree of restriction (Figure 1). Although early-onset of
the CR diet is acceptable in rodents, it would be unreasonable
to apply the same study design in primates. Initiating a CR diet
in young humans or nonhuman primates may negatively
influence development and the attainment of maturity or may
precipitate cognitive defects. Analysis of the effect of adult-
onset CR in mice confirmed that reduced caloric intake
increased average and maximal lifespan in both C3B10F1and
C57Bl/6 strains (Weindruch and Walford 1982), paving the
way for a nonhuman primate study.
EXPLORATION OF THE MECHANISMS OF CR
Numerous hypotheses have been presented to explain the
effect of CR on longevity, including those involving alterations
in membrane and lipid biology, insulin/IGF (insulin-like growth
factor) axis, metabolic rate, systemic inflammation (Heilbronn
and Ravussin 2003; Koubova and Guarente 2003; Masoro 2002;
Smith et al. 2004; Weindruch et al. 2008). The inverse relation-
ship between caloric intake and lifespan suggests that factors
involved in regulation of energy metabolism may be key regu-
lators of CR. A number of nutrient-sensitive proteins that have
been implicated in longevity, including the sirtuins (Bordone
and Guarente 2005), forkhead transcription factors (Greer and
Brunet 2005), and the metabolic regulator mTOR (Schieke and
Finkel 2006), may also be important in CR. These potential
longevity pathways are most likely not mutually exclusive. We
have proposed a model where CR induces an active response to
altered nutrient availability and propose that metabolic repro-
gramming is a primary event in the mechanism of CR (Anderson
and Weindruch 2007) (Figure 2).
Through the application of gene expression profiling, it has
become possible to determine the signature of the biological
age of a tissue at the molecular level. This technique also pro-
vides a means to uncover mechanisms of aging and its retarda-
tion by CR. To explore the effects of age and CR on gene
regulation, we compared transcription profiles in tissues from
young (five months old), old (thirty months old), and old CR
mice (Lee et al. 2002; Lee et al. 1999; Lee et al. 2000). Our
studies have focused on postmitotic tissues, as these are most
vulnerable to the effects of age. In these experiments, the con-
trol mice are mildly restricted (approximately 90% of ad libi-
tum levels) to prevent obesity and control calorie intake;
caloric intake for the restricted animals is reduced by a further
25% (approximately 67% of ad libitum levels). Our investiga-
tions of CR-dependent changes in transcription profiles in aged
mice have revealed two types of transcriptional effects: first,
the CR-induced prevention of age-associated changes and
second, the changes associated with CR that are not age
dependent, that is, those that are indicative of CR-induced tran-
scriptional reprogramming. In the heart, aging is associated
with transcriptional changes that are consistent with increased
structural protein turnover and neurodegeneration in addition
to a shift from fatty acid to glucose metabolism (as illustrated
in Lee et al. 2002). CR opposes the development of many of the
age-associated changes and, importantly, was associated with
FIGURE 1.—Inverse linear relationship between caloric intake and
lifespan in mice. C3B10F1females were individually housed and placed
on controlled diets (115 kcal/week, 85 kcal/week, 50 kcal/week, and
40 kcal/week) from weaning (n = 49, 57, 71, and 60, respectively).
Animals with the highest caloric intake died earliest, and survival
improved with increased caloric restriction.
FIGURE 2.—CR-induced reprogramming of energy metabolism through
activation of master regulators. This metabolic reprogramming results
in an altered metabolic state that positively influences tissue-specific
effectors of longevity pathways, leading to a reduced rate of aging.
Master regulators may include the transcriptional co-activator PGC-1α
and members of the nuclear receptor family.
increased expression of genes involved in energy metabolism
and reduced expression of genes involved in inflammation (Lee
et al. 2002). Regulation of metabolism emerged as an impor-
tant theme from these studies and pointed to mitochondrial
alterations as a critical feature of CR.
Our focus on the role of metabolism in aging led us to inves-
tigate the effect of CR on white adipose tissue (WAT). In recent
times, there has been a considerable revision of the view of adi-
pose tissue as a metabolically inert fat storage depot. It has
become clear that adipose tissue is not only metabolically active,
but it is also a source of hormones and inflammatory factors
that influence metabolic homeostasis and systemic inflamma-
tion (Ahima 2006; Kershaw and Flier 2004; Mohamed-Ali et al.
1998). In order to determine the effect of CR independent of
age-associated changes, we conducted a study in young adult,
age-matched C57B1/6 male mice and compared the effect of
fasting, short-term CR (twenty-three days) and long-term CR
(nine months) on WAT (Higami et al. 2004; Higami et al.
2006). Long-term CR had profound effects on the morphology,
adiposity, and transcriptional profile of WAT. These changes
were not observed with fasting and short-term CR, regimens
that are not associated with increased lifespan. Metabolic
reprogramming is a prominent feature of the CR effect in WAT,
with increased expression of genes involved in the glycolytic
pathway, the lipolytic pathway, amino acid metabolism, and
mitochondrial metabolism (Higami et al. 2004). In addition
to this striking activation of energy metabolism, there was a
marked reduction in the expression of genes involved in
inflammation in WAT (Higami et al. 2006). These alterations in
WAT may provide an explanation for how CR delays the onset
of age-associated diseases that have a basis in systemic inflam-
mation and points to a potential role for WAT-derived factors in
the aging process.
PGC-1α: A MASTER REGULATOR OF
Based on the transcriptional data,we sought to identify factors
that could be responsible for the metabolic reprogramming
induced with CR. The transcriptional co-activator PGC-1α is a
key regulator of genes involved in mitochondrial metabolism,
including the nuclear-encoded genes involved in the electron
transport system (Finck and Kelly 2006; Scarpulla 2006). CR
increases mRNA levels of PGC-1α in multiple tissues (Nisoli
et al. 2005), and PGC-1α transcripts are elevated in cultured
cells treated with serum from CR animals (Lopez-Lluch et al.
2006). In addition to regulating the genes involved in energy
metabolism, PGC-1α also influences the balance between
carbohydrate and fat metabolism through co-activation of the
nuclear receptor family of transcription factors (Corton and
Brown-Borg 2005; Puigserver and Spiegelman 2003).
We recently identified a novel mechanism of PGC-1α regula-
tion in the stress response in cultured cells (Anderson et al.
2008). Mitochondrial energy metabolism is regulated in the early
response to oxidative stress in a PGC-1α−dependent manner,
and there is an increase in mitochondrial membrane poten-
tial. Following stress, PGC-1α localization and transcriptional
activity are regulated by SIRT1-dependent deacetylation. PGC-1α
moves to the nucleus and activates transcription of ETS genes.
The nutrient-sensitive kinase glycogen synthase kinase 3 beta
(GSK3β) is activated by stress and regulates PGC-1α protein
stability by targeting it for proteasomal degradation in the
nucleus. As a result, the nuclear pool of PGC-1α is degraded,
preventing continued activation of transcription, and the cytoso-
lic pool of PGC-1α is replenished, re-establishing the prestress
PGC-1α cellular distribution. This mechanism permits temporal
regulation of mitochondrial function through alterations in
PGC-1a stability, localization, and activity (Figure 3). Analysis
of tissues from mice under conditions of oxidative stress and from
CR mice indicates that this pathway plays a role in the adaptive
response in mice in vivo, and that regulation of mitochondria
through manipulation of SIRT1 and GSK3β is a common feature
of CR and the stress response (Anderson et al. 2008). These
findings identify a potential link between longevity and stress
resistance and may explain why regimens that extend lifespan
often also confer increased stress resistance.
MONKEY CR STUDIES AND IMPLICATIONS FOR HUMAN HEALTH
The positive effect of CR on lifespan has been documented
in multiple species, from yeast to mammals, but has yet to be
demonstrated in a primate species. To determine whether
CR could attenuate rates of change of most biological indica-
tors of aging, including increased “health span” and increased
longevity, a rhesus monkey study was initiated in 1989 at the
Wisconsin National Primate Research Center at the University
of Wisconsin (Kemnitz et al. 1993). Applying a model of adult-
onset CR (approximately 30% restricted), subjects were moni-
tored for food intake before being randomized into control and
CR groups. The overall goals of this study were to develop the
rhesus monkey as a model for the study of aging and to deter-
mine the influence of CR on disease patterns and longevity. In
addition to the expected reduction in body weight, a number of
phenotypes have emerged in the restricted cohort that indicate
that CR is also efficacious in primates. Similar to the rodent
studies, CR animals show a reduction in adiposity (Colman et al.
1998), increased insulin sensitivity (Gresl et al. 2003), and
reduced levels of oxidative damage (Zainal et al. 2000). In
addition, we have recently observed attenuation in the develop-
ment of sarcopenia in the restricted animals (Colman et al.
2008). Preservation of skeletal muscle function may play a role
in preventing age-associated increases in insulin resistance. To
date, the CR monkeys are protected from glucoregulatory
disorders and the symptoms associated with type 2 diabetes. In
addition, the restricted animals have improved cardiovascular
profiles, including reduced levels of CRP (C-reactive protein)
and decreased levels of triglyceride and phospholipids associ-
ated with low-density lipoproteins (Edwards et al. 1998).
Long-term studies exploring the effect of CR on human
lifespan may be beyond reasonable grasp; however, there is
limited information on the effect of CR on humans from popu-
lation studies and from short-term studies. Although reduced
caloric intake is prevalent in numerous global populations, the
effects of malnutrition often overshadow any benefits of CR.
Vol. 37, No. 1, January 2009 CALORIC RESTRICTION AND MAMMALIAN AGING49
An exception is the relatively large proportion of centagenari-
ans in the Okinawan population. Caloric intake is reduced in
the island population compared to that of Japan as a whole
(Chan et al. 1997; Kagawa 1978). Increased longevity among
these people has been taken as evidence that reduced caloric
intake increases average lifespan. In contrast, there is increas-
ing evidence that overindulgence and the resulting obesity
accelerate the onset of numerous disorders previously associ-
ated with aging, including diabetes, hypertension, athero-
sclerosis, and cancer (Das et al. 2004; Wisse 2004). Short-term
studies of CR have confirmed a positive health effect in people
(Heilbronn et al. 2006), although feasibility and compliance
issues arise in studies of longer duration (Racette et al. 2006).
In individuals who voluntarily restrict themselves, CR proved
highly effective in protecting against risk factors associated
with atherosclerosis (Fontana et al. 2004).
The promise of retardation of human aging brings renewed
focus to the biology of aging in mammals and the elucidation
of the mechanistic basis of aging retardation by CR. Although
improved health and increased longevity are enormously
attractive to most people, it seems unlikely that people would
find the idea of reduced caloric intake sufficiently attractive to
actually undertake the regimen. As a result, the development of
nutraceuticals that mimic the effects of CR without requiring a
severe limitation of calorie intake is an area of active investiga-
tion (Barger, Kayo, Pugh et al. 2008; Barger, Kayo, Vann et al.
This work was supported by NIH funding.
Ahima, R. S. (2006). Adipose tissue as an endocrine organ. Obesity (Silver
Spring) 14 (Suppl 5), 242S–249S.
Anderson, R. M., Barger, J. L., Edwards, M. G., Braun, K. H., O’Connor, C. E.,
Prolla,T. A.,and Weindruch,R. (2008). Dynamic regulation of PGC-1alpha
localization and turnover implicates mitochondrial adaptation in calorie
restriction and the stress response. Aging Cell 7, 101–11.
Anderson, R. M., and Weindruch, R. (2007). Metabolic reprogramming in
dietary restriction. Interdiscip Top Gerontol 35, 18–38.
Barger, J. L., Kayo, T., Pugh, T. D., Prolla, T. A., and Weindruch, R. (2008).
Short-term consumption of a resveratrol-containing nutracuetical mixture
mimics gene expression of long-term caloric restriction in mouse heart.
Exp Geront (epub ahead of print).
Barger, J. L., Kayo,T.,Vann, J. M.,Arias, E. B.,Wang, J., Hacker,T. A.,Wang,Y.,
Raederstorff, D., Morrow, J. D., Leeuwenburgh, C., Allison, D. B.,
Saupe, K. W., Cartee, G. D., Weindruch, R., and Prolla, T. A. (2008).
A low dose of dietary resveratrol partially mimics caloric restriction and
retards aging parameters in mice. PLoS ONE 3, e2264.
Bordone, L., and Guarente, L. (2005). Calorie restriction, SIRT1 and metabo-
lism: understanding longevity. Nat Rev Mol Cell Biol 6, 298–305.
Chan, Y. C., Suzuki, M., and Yamamoto, S. (1997). Dietary, anthropometric,
hematological and biochemical assessment of the nutritional status of
centenarians and elderly people in Okinawa, Japan. J Am Coll Nutr
Colman, R. J., Beasley, T. M., Allison, D. B., and Weindruch, R. (2008).
Attenuation of sarcopenia by dietary restriction in rhesus monkeys.
J Gerontol A Biol Sci Med Sci 63, 556–59.
50ANDERSON ET AL.TOXICOLOGIC PATHOLOGY
FIGURE 3.—Alterations in PGC-1α activity and stability through SIRT1 and GSK3β. In response to acute stress, activation of SIRT1 and GSK3β
causes activation and subsequent degradation of PGC-1α, resulting in a rapid and transient effect on mitochondrial energy metabolism. In response
to CR, a chronic stress, SIRT1 is activated and GSK3β is inhibited, causing increased PGC-1α activity and stability, resulting in a prolonged
effect on mitochondrial energy metabolism.