Dossier: Aging and age-related diseases

The anti-ageing effects of caloric restriction may involve

stimulation of macroautophagy and lysosomal degradation,
and can be intensied pharmacologically
E. Bergamini *, G. Cavallini, A. Donati, Z. Gori
Centro di Ricerca di Biologia e Patologia dellInvecchiamento, University of Pisa, Scuola Medica, Via Roma 55, 56126 Pisa, Italy
Abstract
Caloric restriction (CR) and a reduced growth hormone (GH)insulin-like growth factor (IGF-1) axis are associated with an extension of
lifespan across taxa. Evidence is reviewed showing that CR and reduced insulin of GHIGF-1 axis may exhibit their effects at least partly by
their common stimulatory action on autophagy, the cell repair mechanism responsible for the housekeeping of cell membranes and organelles
including the free radical generators peroxisomes and mitochondria. It is shown that the life-long weekly administration of an anti-lipolytic
drug may decrease glucose and insulin levels and stimulate autophagy and intensify anti-ageing effects of submaximal CR.
2003 ditions scientiques et mdicales Elsevier SAS. All rights reserved.
Keywords: Ageing; Autophagy; Caloric restriction
1. Ageing and senescence
The postreproductive phase of life of virtually all multi-
cellular species is characterized by an inexorable, progres-
sive decrease in physiological capacity and reduced ability to
respond to environmental stresses, that lead to diminished
homeostasis and increased organismic vulnerability to dis-
ease [1,2]. The nature of the causal mechanisms that initiate
the deleterious alterations underlying this phenomenon, of-
ten referred to as senescence or the aging process remain
controversial. A currently popular hypothesis postulates that
a progressive accumulation of macromolecular oxidative
damage and not completed housekeeping is the fundamental
underlying cause of senescence-associated deleterious alter-
ations [1]. Oxidative stress is involved in ageing and many
acute and chronic diseases as a major pathogenic factor [3].
May be, in this nite, imperfect world nothing is perfect,
including health. In this perspective, the rate of the process of
ageing of any individual species may be dictated by the gap
between real life and perfection, and senescence could be
dened as a chronic, degenerative, not curable disease with a
life-long incubation time, and age-associated diseases might
be viewed as complications. This is an optimistic view, which
implies that ageing though not curable might be preventable
like any other disease. Hypothesis is in line with current
knowledge: ageing appears to be a phenotypic event like any
other disease, which is inuenced by genetic and environ-
mental factors; interventions in genetic and nutritional fac-
tors were found to extend longevity. Evidence will be re-
viewed showing that both types of approach may act by the
same pathophysiological mechanism, and that the benecial
effects may be intensied pharmacologically.
2. The genetic control of longevity
Genes exert a powerful control on lifespan, as indicated by
the enormous difference among species, ranging from a few
days to more than 100 years. In mammals, genetic factors
account for only about 35% of the intraspecies variance in
longevity [4]. Arapidly growing body of evidence shows that
at least some of the mechanisms of genetic control of longev-
ity might have been conserved from yeast to mammals and
that insulininsulin-like growth factor (IGF) signalling path-
way is causally linked to ageing across taxa [5]. The
insulin/IGF-1 signalling pathway (where IGF-1 is insulin-
like growth factor-1) inuences longevity (acting during
adulthood to relatively old age), reproduction and diapause in
many organisms [6]. In worms, ies and in the transgenic
mice, mutations in the insulin signalling pathway reduce
* Corresponding author.
E-mail address: ebergami@ipg.med.unipi.it (E. Bergamini).
Biomedicine & Pharmacotherapy 57 (2003) 203208
www.elsevier.com/locate/biopha
2003 ditions scientiques et mdicales Elsevier SAS. All rights reserved.
DOI: 10.1016/S0753-3322(03)00048-9
growth and size of the organism and, curiously, prolong
longevity [7,8]. Studies in C. elegans demonstrate that dis-
ruption of the daf-2 signalling pathway extends longevity
[9,10]; similarities with the insulin-like signalling in ies and
yeast, and mammalian insulin-like growth factor I (IGF-1)
signalling cascade raise the possibility that modications to
the IGF-I signalling could also extend lifespan in mammals
[11]. Long-standing growth hormone (GH)/IGF-1 deciency
affects several parameters of the ageing process without
impairing lifespan, and prolongs longevity in animal models
[12], whereas high GH/IGF-1 levels accelerate death [13].
GH/IGF-I decient dwarf mice do live signicantly longer
than their wild counterparts [12]. Heterozygous knockout
Igf1r (+/) mice are not dwarf and live on the average 26%
longer than their wild-type littermates [14], but an optimal
level of hormone appears to be required to maximize survival
[15]. On the other hand, transgenic mice that overexpress GH
exhibit a drastic reduction in lifespan [16]. In invertebrates
and mammals, insulin and IGFs use similar signalling path-
ways and both are importantly related to the regulation of
ageing and lifespan and may interact with each other [17].
Ageing may impair insulin and IGF-1 signalling but does not
affect IGF-1 signalling in hepatic tissues [18].
3. Nutritional intervention in ageing: the effects of CR
Caloric restriction (CR) has been documented to have a
positive effect on the lifespan of rodents and various inverte-
brate speciesprotozoa, ies, water eas, nematodes, roti-
fers and spidersand vertebrates speciessh, hamsters,
dogs [19,20]. In fact, research spanning more than 60 years
has shown that diet restriction is the only nutritional interven-
tion that consistently extends the median and maximum
lifespan and health span of animals [19,21]. There are ongo-
ing long-term studies on the effects of CR on non-human
primates in several US laboratories but none of these studies
has been carried on long enough to yield information on the
effect of restriction on longevity of these monkeys thus far
[22]. Nutrition has remarkable effects on glucose, lipid and
amino acid metabolism and inuences many endocrine func-
tions. Restriction of caloric intake is known to reduce blood
sugar and insulin levels and to lower resistance to insulin of
peripheral tissues [19,23]. Recent evidence invited to suggest
that CR might exhibit its effects to lifespan extension partly
through the reduced GHIGF-1 axis.
4. Mechanisms of the genetic and nutritional
anti-ageing intervention
It has been shown that disruption of the insulin receptor in
the adipose tissue (FIRKO mice) extends longevity, shrinks
mice fat pad and causes a percent wise increase in lean body
mass, and does not decrease (it may rather increase) energy
expenditure per unit of body weight [24]. To our knowledge,
the in vivo administration or the in vitro treatment with
insulin never caused any sudden increase in free radical
production, which could account for a direct pro-ageing
effect of the hormone. CRextends median and maximumlife
and health span, lowers body weight and causes a percent
wise increase in lean body mass; careful physiological stud-
ies showed that it does not cause any decrease in the energy
expenditure per unit of body mass [25]. It was shown that
restriction of the caloric intake for 4 months does not de-
crease oxygen-free radical production [26]. On the whole,
there is no evidence to support the hypothesis that genetic
and nutritional intervention might extend longevity by de-
creasing oxidative metabolism and free radical generation.
Therefore, the alternate hypothesis should be considered that
anti-ageing interventions might extend life and health span
by improving the function of the anti-ageing cell repair
mechanism(s) (Table 1). There is no evidence that the cell
repair mechanisms acting at the molecular level and apopto-
sis are primarily controlled by nutrition. Macroautophagy,
which has not been given much attention as an anti-ageing
cell repair mechanism so far, was proposed to be the best
candidate-mediator of the anti-ageing effects of CR [27,28],
because it is tightly coupled with nutrition and is inhibited by
higher levels of insulin. Macroautophagy has the function to
serve as an important source of amino acids for gluconeogen-
esis and other systemic oxidative and biosynthetic reactions
when exogenous substrate is not available [29,30].
5. Macroautophagy
Macroautophagy is a universal, highly conserved process,
which takes place in all eukaryotic cells. The process is
important to maintain a well-controlled balance between
anabolism and catabolism in order to have normal cell
growth and development. It plays an essential role during
Table 1
Anti-ageing cell repair mechanisms
Mechanism Predictable effects of
malfunctioning
Direct regulation by
nutrition
Molecular level
DNA repair Accumulation of altered
DNA
Not proved
Proteolysis Accumulation of altered
protein
Not proved (proteasomal)
Yes (autophagic-lysosomal)
Fatty acid
replacement
Changes in phospholipid Not proved
Subcellular level
Autophagy Abnormal accumulation of
membrane lipids (dolichol);
accumulation of altered
organelles (mitochondria,
peroxisomes) and increased
oxidative stress
Yes (stimulated by lower
amino acids, insulin, IGF-1
plasma levels)
Cell and tissue level
Apoptosis Accumulation of defective
cells in tissues
Not proved
204 E. Bergamini et al. / Biomedicine & Pharmacotherapy 57 (2003) 203208
starvation, cellular differentiation, cell death and ageing but
also in preventing some form of cancer [31]. This degrada-
tion pathway permits the cell to eliminate unwanted or un-
necessary organelles and to recycle the components for re-
use [32]. The basic machinery for autophagy is identical
between yeast and mammalian cells [33]. Macroautophagy
has also been demonstrated in plants and shown to be in-
volved in the response of plant cells to the shortage of
nutrients [34,35]. Autophagy is involved in cell re-modelling
and atrophy, and is the only mechanism for the degradation
of altered membranes and cell organelles, including mito-
chondria (e.g. [36]). Defective autophagy may result in cell
loss by type II apoptosis. Autophagy is a dynamic process
involving the rearrangement of subcellular membranes to
sequester cytoplasm and organelles for delivery to the lyso-
some or vacuole, where the sequestered cargo is degraded
and recycled. The process is highly regulated through the
action of various kinases, phosphatases and guanosine triph-
osphatases [31]. Extensive research on mammalian cells in
the last three decades showed that autophagy and lysosomal
proteolysis are activated by lower amino acid and insulin
levels during fasting to produce nutrients from endogenous
sources, and that higher (postprandial) levels of insulin fully
suppress autophagy in the physiological range of plasma
amino acid concentration [29,30,37]. In conclusion, it is
conceivable that mammalian cells can use autophagy in order
to get rid of altered membranes and cell organelles only
during the time of organismic fasting.
6. Age-changes in the function of macroautophagy. The
effects of CR
Lysosomal function(s) decline(s) in older animals [38]. In
liver cells isolated from ad libitum fed rats, the function of
autophagy declines with increasing age and the decline is
prevented by CR [39,40]. The ageing-related changes in the
function of autophagy correlate with the expectation of life
[41]. The action of autophagy in vivo becomes weaker and
weaker the older the animal starting by age 6 months in ad
libitum fed animals, but retains its strength in food-restricted
rats [4244]. As expected, the ageing-related decline in au-
tophagy may produce a fall in the maintenance of cell mem-
branes: the lipid composition of rat tissues changes after the
age of 6 months, because of a progressive age-related accu-
mulation of the membrane lipid dolichol [45]. Rats on a
caloric restricted regimen consume their given food in less
than 68 h and spend most of the day in a state of fasting,
lower concentration of glucose and insulin and higher plasma
concentration of leucine, isoleucine and valine and higher
rate of autophagic proteolysis. The ageing-related accumula-
tion of dolichol is signicantly retarded by calorie restriction
[46], efcacy depending on level [47] and duration [48]. The
age-changes in the lipid composition of cytomembranes in
calorie restricted rats correlate with the function of autoph-
agy and expectation of life [41,48].
7. Effects of pharmacological intervention in autophagy
on the rate of ageing
Pharmacological modulation of the autophagic function
may either accelerate or retard ageing. Autophagic-
lysosomal function can be reduced by the injection of inhibi-
tors of thiol proteases (e.g. leupeptin) and lysosomotropic
agents like chloroquine, which are general lysosomal en-
zyme inhibitors [49]. Chronic pharmacological inhibition of
autophagic proteolysis appears to accelerate the rate of the
process of aging: in several species and organ systems, the
treatment induced the formation of lysosomal aggregates
which closely resembled ceroid-lipofuscin [50], which
strongly suggested that the protease-inhibitor model of age-
ing is generally valid [51]. Protease inhibition caused an
anomalous accumulation of tau and ubiquitin immunoreac-
tivity in brain [52]. Function of autophagic proteolysis can
easily be intensied in vivo by a pharmacological interven-
tion on the physiological regulatory mechanism. The admin-
istration of anti-lipolytic agents to fasted rats may provide a
convenient (i.e. an inexpensive, highly reproducible and ti-
mable) physiologic model to study hormone (low insulin
high glucagon and corticosterone)-induced autophagy in
liver cells [53,54]. Treatment caused a signicant degrada-
tion of selected liver cell organelles including peroxisomes
and, to a minor extent, mitochondria [55]. Preliminary results
showed that life-long weekly stimulation of autophagy by the
Fig. 1. Effects of ageing, CR and pharmacological intervention on the
regulatory effect of amino acids on the autophagic proteolysis of liver cells.
Data are given as the difference between the rate of proteolysis in the
absence of amino acid and in the presence of a maximally active amino acid
concentration in the incubation medium(see Refs. [40,41] for details). Liver
donors were C, 2 month old rats; AL, 24 month old rats fed ad libitum; DF,
24 month old rats fasted 1 d a week starting by age 2 months; DA, 24 month
old rats fasted like DF rats and given an anti-lipolytic drug on the day of
fasting; EOD, 24 month old rats on a severe anti-ageing CR(every-other-day
ad libitum) starting by age 2 months (from Ref. [57] with permission). The
effects of age, diet and drug were highly signicant.
205 E. Bergamini et al. / Biomedicine & Pharmacotherapy 57 (2003) 203208
intragastric administration of an anti-lipolytic agent licensed
for human use maximized the benecial effects of a mild
(one-day-a week, 10%) calorie restriction on two parameters
that are known to correlate with life expectancy: the ageing-
related changes in liver autophagy (Fig. 1) and dolichol
accumulation in the liver (Fig. 2) [56].
8. Conclusions
Evidence in ad libitumfed animals suggests that overfeed-
ing may increase plasma glucose and insulin secretion and
cause a long-lasting increase in amino acid, insulin and
IGF-1 plasma levels. Physiological expectation is that the
afore mentioned metabolic and endocrine changes could
suppress autophagy and slow down turnover rate of cell
protein, membrane and organelles. Longer biological life of
cell macromolecules and structures may give more time for
alteration and accumulation of peroxidized macromolecules
and defective organelles in cells [57], and may accelerate
aging. A vicious circle may be started by the alteration of
membrane function, which eventually leads to irreversibility
of the cell changes (see Fig. 3 and Refs. [28,29]). CR and
genetic disruptions of the insulin and GH/IGF-1 axis may
lower insulin and IGF-1 levels and break the cycle: enhanced
autophagy can prevent the age-related accumulation of dete-
riorated subcellular components (including altered mito-
chondria) and extend lifespan. Anti-lipolytic drugs and se-
vere atrophy of the fat tissue (e.g. by genetic disruption of the
fat insulin receptor [24]) may cause a shortage of FFA and a
compensatory intensication of autophagic proteolysis
[54,55] and glucogenesis during fasting, which may account
for benecial effects on longevity.
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