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IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice

  • French National Centre for Scientific Research, Ecole Normale Supérieure de Paris, Paris, France

Abstract and Figures

Studies in invertebrates have led to the identification of a number of genes that regulate lifespan, some of which encode components of the insulin or insulin-like signalling pathways. Examples include the related tyrosine kinase receptors InR (Drosophila melanogaster) and DAF-2 (Caenorhabditis elegans) that are homologues of the mammalian insulin-like growth factor type 1 receptor (IGF-1R). To investigate whether IGF-1R also controls longevity in mammals, we inactivated the IGF-1R gene in mice (Igf1r). Here, using heterozygous knockout mice because null mutants are not viable, we report that Igf1r(+/-) mice live on average 26% longer than their wild-type littermates (P < 0.02). Female Igf1r(+/-) mice live 33% longer than wild-type females (P < 0.001), whereas the equivalent male mice show an increase in lifespan of 16%, which is not statistically significant. Long-lived Igf1r(+/-) mice do not develop dwarfism, their energy metabolism is normal, and their nutrient uptake, physical activity, fertility and reproduction are unaffected. The Igf1r(+/-) mice display greater resistance to oxidative stress, a known determinant of ageing. These results indicate that the IGF-1 receptor may be a central regulator of mammalian lifespan.
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Supplementary Information accompanies the paper on Natures website
Acknowledgements We thank D. Reinberg for the anti-RPB1 antibody; V. Sartorelli for the p300
expression construct; U. Schibler for Rev-Erb
reagents; H. R. Ueda for PATSER sequence
analysis; and D. R. Weaver and C. L. Peterson for suggestions. This work was supported by grants
from the NIH and the Defense Advanced Research Projects Agency (DARPA).
Competing interests statement The authors declare that they have no competing financial
Correspondence and requests for materials should be addressed to S.M.R.
IGF-1 receptor regulates lifespan and
resistance to oxidative stress in mice
Martin Holzenberger*, Joe
lle Dupont, Bertrand Ducos*,
Patricia Leneuve*, Alain Ge
n, Patrick C. Even§, Pascale Cerverak
& Yves Le Bouc*
* Institut National de la Sante
et de la Recherche Me
dicale U515, and kService
d’Anatomie et de Cytologie Pathologiques, Ho
pital Saint-Antoine, 75571 Paris 12,
Institut National de la Recherche Agronomique, 37380 Nouzilly, France
Institut National de la Sante
et de la Recherche Me
dicale U352, INSA, 69621
Villeurbanne, France
§ Institut National de la Recherche Agronomique, INA P-G, 75231 Paris 5, France
Studies in invertebrates have led to the identification of a number
of genes that regulate lifespan, some of which encode com-
ponents of the insulin or insulin-like signalling pathways
(Drosophila melanogaster) and DAF-2 (Caenorhabditis elegans)
that are homologues of the mammalian insulin-like growth
factor type 1 receptor (IGF-1R). To investigate whether IGF-1R
also controls longevity in mammals, we inactivated the IGF-1R
gene in mice (Igf1r). Here, using heterozygous knockout
mice because null mutants are not viable, we report that
mice live on average 26% longer than their wild-type
littermates (P < 0.02). Female Igf1r
mice live 33% longer than
wild-type females (P < 0.001), whereas the equivalent male
mice show an increase in lifespan of 16%, which is not statisti-
cally significant. Long-lived Igf1r
mice do not develop dwarf-
ism, their energy metabolism is normal, and their nutrient
uptake, physical activity, fertility and reproduction are un-
affected. The Igf1r
mice display greater resistance to oxi-
dative stress, a known determinant of ageing. These results
indicate that the IGF-1 receptor may be a central regulator of
mammalian lifespan.
Insulin/insulin-like signalling molecules that have been linked to
longevity include DAF-2 and InR, and inactivation of the corre-
sponding genes leads to increased lifespan in nematodes
, respectively. Null mutations of the insect insulin-receptor
substrate Chico, which acts downstream from InR, also extends
. Most long-lived daf-2 and Inr mutants develop dwarfism
and hypofertility; however, some Inr mutants display normal
fertility and growth. This suggests that longevity may be regulated
independently of body size and reproduction
. DAF-2 and InR are
structural homologues of a family of vertebrate tyrosine kinase
receptors that includes the insulin receptor and the insulin-like
growth factor type 1 receptor (IGF-1R). In vertebrates, the insulin
receptor regulates energy metabolism
whereas IGF-1R promotes
. IGF-1R is activated by its ligand IGF-I, which is secreted in
response to growth hormone. It is unclear whether the insulin
receptor or IGF-1R, or both, have taken over responsibility for
lifespan regulation in vertebrates
. The phenotypes of long-lived
spontaneous mouse mutants studied so far indicate a probable link
between longevity and growth. The long-lived Prop1
dwarf) and Pit1
(Snell dwarf) mutants
display impaired
pituitary gland development and low levels of pituitary hormones,
including growth hormone. These mutants are sterile dwarfs. The
recent targeted inactivation of the growth hormone receptor itself,
Figure 1 IGF-1R gene targeting, receptor expression and growth phenotype. a,We
flanked exon 3 of the wild-type (WT) IGF-1R gene with a neomycin-resistance cassette
) and two loxP sites (triangles). Exon 3 and neo
were then deleted by Cre-lox
recombination, producing the Igf1r
(knockout) allele
. b, Allele-specific RT–PCR
revealed that heterozygous Igf1r
mice produced mRNA from wild-type (þ) and
knockout (2) alleles (double band in lanes 2 and 3). M, DNA size marker. c, Although
levels of IGF-1R were halved in Igf1r
animals (bar graph), the relative distributions
(autoradiographic pattern) were unchanged. Receptors were undetectable in Igf1r
embryos (data not shown). Together, this indicates that the remaining, intact allele does
not compensate for its inactivated homologue. Nonspecific binding was 8%. Scale bar,
5 mm. Asterisk, P , 0.05; double asterisk, P , 0.01 (Mann-Whitney U-test). See also
Supplementary Information. d, Igf1r
and wild-type siblings show identical growth
until day 20. Thereafter, during the prepubescent growth spurt (weeks 3–5), slight deficits
appear. Asterisk, P , 0.05 (Mann–Whitney U-test).
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NATURE | VOL 421 | 9 JANUARY 2003 |
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which strongly decreases circulating IGF-I and impairs growth and
development, also increases lifespan
. Similarly, caloric restriction,
the only efficient treatment known to increase mammalian lifespan,
invariably reduces circulating IGF-I levels, and, if begun in juveniles,
also engenders dwarfism. These findings led us to investigate
whether mammalian lifespan is regulated by IGF-1R.
We inactivated the IGF-1R gene by homologous recombination
using the Cre-lox strategy to delete the essential exon 3 of the
(Fig. 1a). We found that homozygous null mutants (Igf1r
) died at birth, as previously described in a study using classical
insertional mutagenesis
. Our Igf1r
mutant transmitted the
null allele in the expected mendelian ratio (52%, n ¼ 241). Wild-
type and Igf1r-null transcripts were present (Fig. 1b); however, as
Igf1r-null transcripts cannot be translated into functional protein
IGF-1 receptor levels in Igf1r
mice were half those in wild type
mice (Fig. 1c). Weight at birth and during the first three
weeks of growth were nevertheless normal (Fig. 1d). Only after the
natural weaning period (around day 20) did Igf1r
males develop
a modest, 8% growth deficit with respect to their Igf1r
mates (23.1 ^ 0.7 g compared with 25.1 ^ 0.7 g at age 7 weeks,
P , 0.05), whereas the growth deficit did not exceed 6% in females
(19.5 ^ 0.6 g compared with 20.7 ^ 0.5 g at 7 weeks, not significant
(NS)). These modest weight differences affected all tissues to similar
degrees, persisted throughout life (data not shown), and resembled
the growth pattern observed in previous models of partial inacti-
vation of the IGF-1R gene
. Thus, the bi-allelically expressed mouse
IGF-1R gene is heteroinsufficient.
Fed ad libitum on a standard diet and maintained in regular
housing until natural death, mice with only one functional IGF-1R
allele significantly outlived their wild-type littermates (Fig. 2).
mice lived a mean of 26% longer than Igf1r
(P , 0.02; Cox’s test). If the sexes were evaluated separately, mutant
females were found to live 33% longer than wild-type females
(P , 0.001), whereas mutant males lived only 15.9% longer than
control males (NS). On average, Igf1r
females outlived Igf1r
males, whereas the opposite is normally the case in wild-type
populations of the 129/J genetic background
(Fig. 2). Thus, in
mice, the degree to which lifespan is extended depends on
sex, as has been described for Drosophila mutants with impaired
insulin-like signalling
. Our ageing cohorts had a 5% tumour
incidence, unrelated to genotype and consistent with the low (7%)
general tumour incidence of mice with the 129/J background
Necropsy revealed a number of different diseases, consistent with
the results of previous studies, showing that mice with this back-
ground do not develop specific age-related diseases
. We did not
observe accidental deaths, although some of the sporadic mortality
of younger males may have been consequences of fights for
Blood parameters (see Methods) were normal. However, serum
IGF-I levels were upregulated in adult Igf1r
mice (males,
795 ^ 64 compared with 625 ^ 30 ng ml
, P , 0.01; females,
716 ^ 39 compared with 516 ^ 14 ng ml
, P , 0.001; n ¼ 8–10
per group) and may reflect an endocrine response to the reduced
availability of IGF-1R
. Blood glucose levels in mice that were
deprived of food overnight were unaffected. In fed animals, how-
ever, Igf1r
males tended to have higher (þ12%) and Igf1r
females to have lower (24.4%) blood glucose levels than the
controls. Non-fasting insulin levels were nevertheless normal
(males, 1.65 ^ 0.12 compared with 1.40 ^ 0.14 ng ml
; females,
1.58 ^ 0.25 compared with 1.90 ^ 0.25 ng ml
; n ¼ 8–10 per
group). We then tested glucose tolerance in mice that were deprived
of food overnight (Fig. 3a, b) and found that Igf1r
males had a
significantly stronger glucose response than controls (P , 0.001).
In Igf1r
females, the response was slightly weaker than in wild-
Figure 2 Lifespan extension in Igf1r
mice with respect to Igf1r
(WT) mice.
a, Igf1r
females (thick line) live a mean of 33% longer than their wild-type littermates
(756 ^ 46 compared with 568 ^ 49 days; P , 0.01, t-test). Kaplan–Meier analysis of
survival revealed a later decline in Igf1r
mice compared with wild type (P , 0.001,
Cox’s test). b, Igf1r
males live 15.9% longer than wild-type littermates (679 ^ 80
compared with 585 ^ 69 days; NS).
Figure 3 Glucose tolerance and energy metabolism in Igf1r
mice. a, After an
intraperitoneal glucose injection, the glucose response is strongest in mutant males. Note
that the significant sex-related dimorphism of the response observed in wild-type mice is
even more marked between mutants. Asterisk, P , 0.05; double asterisk, P , 0.01;
triple asterisk, P , 0.001. b, Combining data from both sexes largely cancels out the
male hyperglycaemic phenotype. Asterisk, P , 0.05; n ¼ 89. c, Metabolic rate,
measured by indirect calorimetry, did not differ between groups whether we determined
mean (24 h), resting or basal metabolic rate (n ¼ 11–12 per group).
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type females (P , 0.05). It remains unclear whether this hypergly-
caemic effect in Igf1r
males is related to the reportedly com-
-cell mass in these mice
As metabolism may have an important role in ageing, we
explored energy expenditure in Igf1r
mice. Body temperature,
indicative of metabolic activity and reported to be low in long-
lived Ames dwarf mice
, was unaffected in Igf1r
mice: skin
surface temperature was 36.1 ^ 0.1 8C for all 4 groups (n ¼ 9–14
per group) and rectum temperature was 37.4 ^ 0.2 compared
with 37.5 ^ 0.2 8Cinmalesand37.7^ 0.2 compared with
37.9 ^ 0.2 8C in females (NS). We analysed physical activity,
using a photoelectric actimeter, and found identical circadian
profiles for Igf1r
and Igf1r
mice (data not shown). Further-
more, as caloric restriction is known to extend rodent lifespan, we
investigated the possibility that Igf1r
mice were able to restrict
their own food intake. We measured short-term (1–3 days) and
long-term (90 days) food intake in adults. We observed only
marginal differences between Igf1r
and Igf1r
mice, with
mean food intake (in g d
body weight) being 190 ^ 2
compared with 204 ^ 4 (NS) in males and 148 ^ 4 compared
with 144 ^ 2 (NS) in females, and mean water intake (in
ml d
body weight) being 249 ^ 7comparedwith
255 ^ 29 (NS) in males and 178 ^ 4 compared with 166 ^ 3
(NS) in females.
As mice may use nutrients with variable efficiency, we determined
their metabolic rates. We obtained similar mean metabolic rates in
fed animals over 24 h (males, 332.8 ^ 6.7 compared with 336.0 ^
8.4 J min
; females, 351.9 ^ 6.0 compared with 337.9 ^
7.0 J min
; NS, n ¼ 11–12 per group) (Fig. 3c). The resting
metabolic rate was also similar between mutants and controls. As
the difference between resting and 24-h metabolic rate depends
mainly on physical activity, these results are consistent with the
observed identical activity profiles. Even basal metabolic rate,
measured in the fasted state, did not differ between mutants and
controls (males, 133.2 ^ 5.3 compared with 132.0 ^ 4.7 J min
; females, 135.8 ^ 9.4 compared with 140.9 ^ 6.6 J min
Long-lived C. elegans daf-2 mutants and long-lived dwarf mice
display changes in fertility. We therefore monitored fertility and
reproduction in Igf1r
female mice from puberty to the age of 13
months. Igf1r
females became fertile at 5.2 ^ 0.1 weeks whereas
femalesbecamefertileat5.6^ 0.3 weeks (5.8 ^ 0.1
compared with 6.2 ^ 0.3 in males; n ¼ 7–11 per group). Although
these differences were not significant, Igf1r
mice became fertile,
Figure 4 Mutants show normal fertility and are resistant to oxidative stress. a, Three-
week mating results in a similar proportion of pregnancies in mutant and wild-type
females. These proportions decline from 5 to 13 months of age (P , 0.001,
b, Although offspring decrease markedly with age, there are no consistent differences
between Igf1r
and control females. c, Oestrus cycle length increases significantly
with age (triple asterisk, P , 0.005), reflecting changes in the hormonal control of
ovarian function, but we observed no differences between genotypes. d, The interval
between the first copulation plug (from a sterile male) and the next, indicative of ovarian
capacity to maintain pseudogestation, decreases significantly with age in both groups.
Asterisk, P , 0.05; double asterisk, P , 0.02; n ¼ 15. e, Oxidative stress is induced by
intraperitoneal paraquat injection (70 mg per kg body mass). We checked the mice every
2 h and censored the test at 72 h. Kaplan–Meier analysis shows significantly more
survivors among Igf1r
mice (P , 0.05, Cox’s test; n ¼ 67). When evaluated
separately (inset), female mutants exhibit increased stress resistance (P ¼ 0.05, log-rank
test; n ¼ 37), whereas the increase in males (n ¼ 30) is small.
Figure 5 Lack of IGF-1R reduces activation of major intracellular signalling pathways in
cultured MEFs. We stimulated MEFs with rhIGF-I (þ) and analysed them by western blot.
a, IGF-1R was reduced in Igf1r
and absent from Igf1r
MEFs. b, Anti-IGF-1R
anti-IRS-1 and anti-p66 Shc immunoprecipitates (IP) were probed with anti-
phosphotyrosine (anti-P-Tyr) antibodies. Phosphorylation of these proteins was reduced in
and absent from Igf1r
MEFs. A phospho-Shc antibody revealed reduced
activation of p52 Shc. c, d, IGF-I-induced association of Grb2 with IRS-1 or p52 Shc, and
activation of ERK1/2 MAP kinases and Akt, are also reduced in Igf1r
and absent from
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NATURE | VOL 421 | 9 JANUARY 2003 |
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on average, 3 days earlier than controls. This suggests that IGF-1R
insufficiency does not delay sexual maturation, in contrast to
the growth hormone receptor and binding protein (GHR/BP)
. The litter size of young Igf1r
females was
6.3 ^ 0.5 (n ¼ 7 litters), which is not different from wild-type
129/Sv females (6.4 ^ 0.2 live newborns; n ¼ 120 litters). We
analysed the age-related decline in female fertility, determining
frequency of pregnancies, number of live newborns, mating beha-
viour, oestrus cycle length and ovarian capacity to maintain
pseudogestation (Fig. 4a–d). As expected, fertility decreased mark-
edly with age, but Igf1r
females and their controls displayed
indistinguishable profiles.
Oxidative stress is a principal cause of ageing
, and mouse and fly
mutants with enhanced resistance to oxidative stress are long-
. We therefore subjected adult mice to oxidative stress by
injection of paraquat, a herbicide that induces formation of reactive
oxygen species. Igf1r
mutants resisted this challenge signifi-
cantly longer than controls (Fig. 4e). This increase in stress resist-
ance seemed to be more pronounced in female than in male mutants
(Fig. 4e, inset). To further substantiate these results, we induced
oxidative damage in cultured mouse embryonic fibroblasts (MEFs)
by low concentrations of H
, and found that the proportion of
surviving cells was significantly higher in Igf1r
than in control
MEFs after 24- and 72-h treatments (24 h, 94% ^ 3 compared with
82% ^ 3, P , 0.02;72h,88%^ 3 compared with 68% ^ 2,
P , 0.001).
Mutations of the Drosophila IRS homologue Chico
and of other
proteins acting downstream of IGF-1R, such as mouse p66 Shc
and C. elegans phosphatidylinositol-3-OH kinase (AGE-1)
, but
also regulation of forkhead transcription factor DAF-16 by Akt,
increase lifespan. We therefore investigated how the reduced IGF-1R
levels affected intracellular signalling in this model. We derived
embryonic fibroblasts from wild type, Igf1r
and Igf1r
and studied signalling by western blot. As expected,
cells showed 50% reduction in IGF-1R levels (Fig. 5a).
Immunoprecipitation and western analysis also showed a marked
reduction in IGF-I-induced tyrosine phosphorylation of IGF-1R
and of its substrate IRS-1 (Fig. 5b). The tyrosine phosphorylation
of both p52 and p66 isoforms of Shc, another major substrate of
IGF-1R, were also reduced by half (Fig. 5b). This is of interest as
reduced p66 Shc activation in Igf1r
cells could be a mechanism
by which IGF-I regulates oxidative stress resistance. Shc and IRS-1
bind Grb2 on phosphorylation and thereby activate the mitogen-
activated protein (MAP) kinase pathway, involved in mitogenic
response. The amount of Grb2 co-immunoprecipitating with
p52 Shc or IRS-1 are reduced to half in Igf1r
cells (Fig. 5c).
Two important pathways activated by IGF-I are the MAP kinase
ERK1/2 and phosphatidylinositol-3-OH kinase/Akt kinase signal-
ling cascades. Consistently, IGF-I-stimulated phosphorylation of
ERK1/2 and Akt is reduced by 40–50% in mutant cells (Fig. 5d).
Together, this suggests that IGF-1R heteroinsufficiency downregu-
lates the principal pathways stimulated by IGF-I.
These results show that a general decrease in IGF-1 receptor levels
can increase lifespan in a mammalian species. Thus, the genetic link
between insulin-like signalling and longevity, originally discovered
in non-vertebrates
, also seems to exist in higher vertebrates.
Unlike long-lived mouse mutants with hypopituitarism
with complete lack of GHR/BP
, long-lived Igf1r
mutants, in
which receptor levels were only reduced by 50%, did not develop
dwarfism or hypofertility. We obtained these results using a 129/Sv
genetic background. Preliminary results, however, using an IGF-1R
knockdown mutation
on a hybrid background (129/Sv £ C57Bl/
6J) confirm our findings on lifespan extension (M.H., unpublished
data). IGF-1R is involved in the regulation of carbohydrate metab-
olism and in the pancreatic control of glucose homeostasis
therefore not surprising that we found abnormal regulation of
blood glucose in Igf1r
mice. This abnormality affects only
males and is clearly a sex-related dimorphism. Reduced glucose
tolerance is a symptom of prediabetes, and its potential conse-
quences may have masked an otherwise possibly greater life-
prolonging effect of IGF-1R insufficiency in males. We have
observed previously other gender-related phenotypic differences
in IGF-1R mutants
, and have proposed the interplay of sex-
dimorphic pulsatile growth hormone regulation, paracrine
secretion and signalling of IGF-I, and androgen/oestrogen actions
at the target cell level as possible explanations. Furthermore, similar
sex dimorphism of longevity has been reported in InR mutant
Drosophila and in long-lived Ames dwarf mice
. It is tempting to
speculate on the physiological significance of this sex dimorphism
because major sex differences in lifespan have been found in
numerous species, but clearly additional studies are needed. We
thought that the observed variations in blood glucose might have
had consequences for energy metabolism and expenditure, and that
these mice might present features of caloric restriction. However, we
show that Igf1r
mice have normal food uptake, physical activity
or metabolic rate, which excludes metabolic differences as the cause
of their longevity. It is, on the contrary, possible that the life-
prolonging effects of caloric restriction are due to decreases in
circulating IGF-I levels, mimicking the IGF-1R insufficiency pro-
duced here.
p66 Shc
is the only other targeted mutation in mammals
described so far that leads to a comparable increase in lifespan
without inducing major side effects
. The p66 isoform of Shc
mediates cellular responses to oxidative stress and is, together
with IRS-1, a major cytoplasmic signal transduction molecule for
IGF-1R. Thus, the resistance of Igf1r
mice to oxidative stress is of
considerable interest, and by showing that the stress-regulating p66
Shc is underphosphorylated in IGF-1R deficiency we found a
plausible mechanism connecting IGF signalling to oxidative stress.
Caloric restriction and decreases in the response to oxidative stress
and in insulin-like growth factor signalling all efficiently extend
lifespan in mice. However, it is unclear how these mechanisms
cooperate and the extent to which they are independent. Data from
Drosophila chico
mutants showing that lifespan is extended much
more than can be explained by the modest increase in resistance to
oxidative stress
suggested that the two mechanisms operate inde-
pendently, at least in part, to generate longevity. However, the issue
of cooperation and independence of caloric restriction, insulin-like
signalling and oxidative stress in lifespan extension remains
. Owing to strong oxidative stress-resistance phenotypes
associated with C. elegans longevity mutations
, these aspects merit
further study in vertebrates. Our Igf1r
mutants provide an
invaluable tool for future exploration of the mechanisms of lifespan
regulation. However, it will also be necessary to try to overproduce
IGF antagonists, to administer inhibitors of IGF-1R activation, or to
block signal transduction. It has recently been shown that lifespan
regulation through insulin-like signals in non-vertebrates probably
occurs in a non-cell-autonomous fashion
. Neurons in the central
nervous system, by sensing the circulating levels of ligand, may have
a central function in regulating the ageing of other tissues by means
of hypothetical endocrine mechanisms. We have begun to investi-
gate this possibility in a mammalian model, using the Cre-lox
approach to produce brain-specific IGF-1R knockout mice. Homo-
zygous mice for this mutation are microcephalic, sterile, and have a
complex neuroendocrine dysfunction, but heterozygous mice are
healthy and are useful for lifespan studies. A
The Igf1r mutant, described elsewhere
and available from,
was maintained in the heterozygous state in the 129/Sv genetic background. By mating
males with 9- to 12-week-old 129/Sv wild-type females, we generated three
cohorts, each composed of heterozygous Igf1r
and Igf1r
(wild type) littermates.
Animals lived in conventional conditions: 23 8C, 14/10-h light/dark cycle, standard diet
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(49% carbohydrates, 24% proteins, 5% lipids, 12% humidity, 10% minerals and fibre) and
water ad libitum. We separated mice from mothers on day 30 and grouped them as 6 males
or 6 females per cage, with both genotypes present in each cage. Mice from cohort 1 (20
and 17 Igf1r
females; 12 Igf1r
and 16 Igf1r
males) were checked daily
but otherwise left undisturbed until they died naturally. Single surviving females were
placed in the neighbouring cage, whereas single surviving males received a female for
company. We performed necropsy whenever possible, including tumour
immunohistochemistry. Four animals were killed when death appeared imminent, to
reduce suffering. We drew Kaplan–Meier survival curves using dates of birth and death.
Cohort 2 (9 Igf1r
and 15 Igf1r
females; 14 Igf1r
and 11 Igf1r
males) was
used for blood biochemistry and analysis of glucose tolerance, food consumption, fertility
and body composition. In cohort 3 (17 Igf1r
and 11 Igf1r
females, 14 Igf1r
15 Igf1r
males) we analysed growth, energy expenditure (by indirect calorimetry),
blood parameters, glucose tolerance, and finally in vivo resistance to oxidative stress
induced by methyl viologen (paraquat) injection. We conducted experiments according to
institutional guidelines for care of laboratory animals.
IGF-1 receptor expression
Recombinant human IGF-I (rhIGF-I, for in vitro ligand-binding assays, described
) and rhdes(1-3)IGF-I (for autoradiography) were labelled with
I (see also
Supplementary Information).
For the allele-specific expression assay, we used total RNA from Igf1r
embryos and
triplex reverse transcriptase-polymerase chain reaction (RT–PCR)
. We co-amplified
corresponding fragments from the coding region of the messenger RNA specific for
the wild-type and the inactivated receptor allele. The single forward primer
annealed with exon 2, the first reverse primer
annealed with exon 3, and the second reverse primer
spanned the knockout-specific splice junction
between exons 2 and 4. RT–PCR products of 518 base pairs (bp) corresponded to the
transcript of the knockout allele, and 574-bp products corresponded to wild type. We
extracted total RNA from embryo homogenates by RNAXEL and performed One-Step
RT–PCR using GeneAmp 2400 cyclers. For the reverse transcription reaction, we
incubated 30 ng total RNA at 50 8C for 30 min and at 94 8C for 2 min, followed by 40 PCR
cycles, each consisting of 30-s segments at 94, 59 and 72 8C.
Postnatal growth
To synchronize individual growth, we recomposed 7 litters (cohort 3) on day 1 to yield 8 or
9 pups per mother. We identified newborn mice with coloured ink and permanently
numbered them on day 8. For 11 weeks we weighed them daily at 16:00 on an electronic
balance. Growth curves used sliding means of present weight and weight on the day before
and after.
We determined the onset of male and female fertility by mating Igf1r
and Igf1r
mice from day 30 onwards with fertile wild-type partners (three females per male). The age
of delivery and litter size (when testing females) were compared between genotypes. To
evaluate the decline in female fertility over time we used three sets of parameters.
Measurements started at 5 months of age and were repeated 3 or 4 times at 2-month
intervals. First, we monitored the oestrus cycle by vaginal smear histology for 18 days.
Second, we analysed sexual behaviour and the duration of pseudogestation by mating
females for 2 weeks with vasectomized males and recording vaginal plugs. Third, we mated
females with fertile males for three weeks and recorded the resulting pregnancies and
Blood tests
We measured total bilirubin, cholesterol, creatinine, glucose, lactate, total protein,
triglycerides, urea and uric acid in 5-month-old mice. Circulating IGF-I was measured
using a double-antibody RIA from Diagnostic Systems Laboratories and plasma insulin
using the Linco Sensitive Rat Insulin RIA. We tested glucose tolerance in mice that had
gone without food for 14 h overnight by intraperitoneal injection with 2 g kg
weight of 25%
D-glucose. We measured circulating glucose in tail blood at 0, 15, 30, 60 and
120 min using Lifescan Glucotouch.
Indirect calorimetry
We determined metabolic rate by indirect 24-h calorimetry (see Supplementary
Information for details).
Experiments using MEFs
We established female MEFs from Igf1r
, Igf1r
and Igf1r
embryonic day (E)14
fetuses. IGF signalling pathways and in vitro resistance to H
were studied in early
passages of MEFs. For IGF signalling we removed serum (10% FCS) from MEF cultures
16 h before analysis. Cells were stimulated with 3 nM rhIGF-I (Genentech) for 10 min
before analysis, except for detection of phosphorylated Shc (5 min). For H
we treated MEFs with 100
and determined cell viability after 1–3 days using
Trypan blue and a haemocytometer. n ¼ 6 for each group, in two independent
Western blotting
We performed immunoprecipitation and western blotting as described
Supplementary Information for details).
For group comparisons, we used Student’s t-test. Means are expressed ^ standard error of
the mean (s.e.m.). Error bars represent the s.e.m. We determined the significance of
survival curves by Cox’s test. We used nonparametric Mann–Whitney and
tests where
Received 4 October; accepted 18 November 2002; doi:10.1038/nature01298.
Published online 4 December 2002.
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Supplementary Information accompanies the paper on Natures website
letters to nature
NATURE | VOL 421 | 9 JANUARY 2003 |
© 2003
Acknowledgements We thank P. Monget for contributions to the experimental design;
N. R. Holzenberger for assistance with metabolic and growth studies; G. Hamard for help with
MEFs; J. Sappa for language revision; F. Veinberg for blood biochemistry; and P. Casanovas and
M.-C. Samson for animal care. MENRTsponsored this study with a grant to M.H. and Y.L.B. We
thank D. LeRoith for support to J.D.
Competing interests statement The authors declare that they have no competing financial
Correspondence and requests for materials should be addressed to M.H.
Srb10/Cdk8 regulates yeast
filamentous growth by
phosphorylating the
transcription factor Ste12
Chris Nelson, Susan Goto, Karen Lund, Wesley Hung & Ivan Sadowski
Department of Biochemistry and Molecular Biology, University of British
Columbia, Vancouver, British Columbia V6T 1Z3, Canada
The budding yeast Saccharomyces cerevisiae differentiates into
filamentous invasively growing forms under conditions of nutri-
ent limitation
. This response is dependent on the transcription
factor Ste12 and on the mating pheromone-response mitogen-
activated protein (MAP) kinase cascade
, but a mechanism for
regulation of Ste12 by nutrient limitation has not been defined.
Here we show that Ste12 function in filamentous growth is
regulated by the cyclin-dependent kinase Srb10 (also known as
Cdk8), which is associated with the RNA polymerase II holo-
enzyme. Srb10 inhibits filamentous growth in cells growing in
rich medium by phosphorylating Ste12 and decreasing its sta-
bility. Under conditions of limiting nitrogen, loss of Srb10
protein and kinase activity occurs, with a corresponding loss of
Ste12 phosphorylation. Mutation of the Srb10-dependent phos-
phorylation sites increases pseudohyphal development but has
no effect on the pheromone response of haploid yeast. Srb10
kinase activity is also regulated independently of the mating
pheromone-response pathway. This indicates that Srb10 controls
Ste12 activity for filamentous growth in response to nitrogen
limitation and is consistent with the hypothesis that Srb10
regulates gene-specific activators in response to physiological
signals to coordinate gene expression with growth potential.
Yeast differentiate into elongated invasively growing filamentous
forms to facilitate foraging under conditions of nitrogen or carbon
. This response requires the transcription factors Ste12
and Tec1, which bind cooperatively to filamentous response
elements (FREs) within the promoters of filamentous response
. Transcription from FREs is induced in response to nitrogen
, and filamentous response requires upstream signalling
components of the MAP kinase pheromone-response pathway, but
a mechanism for regulation of Ste12 by nutrient limitation has not
been identified.
Yeast lacking the RNA polymerase II holoenzyme-associated
cyclin-dependent kinase (CDK) Srb10 show increased expression
of Ste12-dependent filamentous responsive genes (ref. 5 and
Fig. 1a), form more extensive pseudohyphae in nitrogen-depleted
(SLAD) medium (Fig. 1b) and show constitutive pseudohyphae
even on rich medium (data not shown). This effect is dependent on
STE12, because diploid yeast with disruptions of both srb10 and
ste12 do not form pseudohyphae on nitrogen-limiting medium
(Fig. 1b, srb10, ste12) and have barely detectable levels of FRE-
dependent transcription (Fig. 1a). Disruption of ste11,which
encodes the pheromone-responsive MAP/extracellular-signal-
regulated kinase (ERK) kinase kinase (MEKK), also prevents
filamentous growth (Fig. 1b) and FRE-dependent transcription
(Fig. 1a) in srb10 yeast. Therefore, elimination of Ste12 activity,
by either gene disruption or impairing basal signalling, prevents the
hyperfilamentous response of yeast lacking Srb10, suggesting that
Srb10 may inhibit filamentous growth by modulating the activity of
Consistent with this possibility, we found that Ste12 is a substrate
for purified recombinant Srb10–Srb11 (Cdk8–cyclinC) complexes
in vitro (Fig. 1c). Wild-type Srb10–Srb11 complexes phosphory-
lated recombinant Ste12 (Fig. 1c, lane 1), but not the inhibitors Dig1
and Dig2 (lanes 3 and 4). By contrast, a mutant Srb10 protein with
impaired kinase activity (Asp290Ala)
only weakly phosphorylated
Ste12 in vitro and also underwent inefficient autophosphorylation
(Fig. 1c, lane 2). Ste12 was phosphorylated on two peptides by Srb10
in vitro (Fig. 1d, peptides 3 and 4); these peptides co-migrated with
phosphopeptides 3 and 4 derived from Ste12 labelled in vivo (ref. 6,
Figure 1 Phosphorylation of Ste12 by Srb10 inhibits filamentous responsive transcription.
a, Deletion of srb10 increases FRE-dependent transcription. Homozygous diploid strains
bearing an FRElacZ reporter
were grown at 30 8C in selective media and assayed for
-galactosidase activity
. b, Hyperfilamentation of srb10 strains requires STE12 and an
intact MAPK cascade. Homozygous diploid yeast were streaked on SLAD plates and
photographed after 3 d. c, Srb10 phosphorylates Ste12 in vitro. Kinase reactions were
carried out with purified recombinant wild-type Srb10–Srb11 (ref. 12; lanes 1, 3, 4) or a
kinase-deficient Asp290Ala Srb10–Srb11 mutant (lane 2) plus recombinant Ste12 (lanes
1, 2), GST–Dig1 (lane 3) or GST–Dig2 (lane 4). Input substrate GST–Dig1 and GST–Dig2
proteins were immunoblotted with antibodies to GST (lanes 5, 6). d, Srb10 phosphorylates
two sites on Ste12. Tryptic phosphopeptide analysis of in vitro phosphorylated Ste12 was
done as described
. The resulting peptides co-migrate with Ste12 phosphopeptides 3 and
4 (not shown). e, Srb10 is required for phosphorylation of Ser 261 and Ser 451 in Ste12
in vivo. Shown is tryptic phosphopeptide analysis of in vivo phosphorylated Ste12
recovered from wild-type or srb10 strains expressing wild-type STE12 (left panels),
S261A STE12 (top right) or S451A STE12 (bottom right).
letters to nature
NATURE | VOL 421 |9 JANUARY 2003 | 187
© 2003
... In mammals, this signaling mechanism begins with insulin binding its receptor tyrosine kinase (insulin/IGF-1 receptor) to induce receptor dimerization and autophosphorylation of tyrosine moieties within the cytoplasm. There is evidence in the literature that CR decreases serum IGF-1 levels in rodents (Holzenberger et al. 2003). Moreover, CR is associated with improved cellular glucose uptake and insulin sensitivity through PPAR-gamma (Zierath et al. 1998;Lee et al. 1999). ...
... In mammalian species, similar results implicate the insulin/IGF-1 pathway as an important lifespan regulator. One group reported that heterozygoteknockout IGF-1 receptor (IGF-1R) mice outlived the wild-type cohorts by approximately 26% when fed a standard isocaloric diet (Holzenberger et al. 2003). Furthermore, the heterozygous IGF-1R mice maintained normal energy metabolism and basal metabolic rate compared to controls. ...
... Furthermore, the heterozygous IGF-1R mice maintained normal energy metabolism and basal metabolic rate compared to controls. Moreover, they displayed more resistance to oxidative stress and showed minimal development of notable age-related pathologies (Holzenberger et al. 2003). However, these beneficial results were exclusive to female heterozygotes. ...
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Over the past decade, extensive efforts have focused on understanding age-associated diseases and how to prolong a healthy lifespan. The induction of dietary protocols such as caloric restriction (CR) and protein restriction (PR) has positively affected a healthy lifespan. These intervention ideas (nutritional protocols) have been the subject of human cohort studies and clinical trials to evaluate their effectiveness in alleviating age-related diseases (such as type II diabetes, cardiovascular disease, obesity, and musculoskeletal fragility) and promoting human longevity. This study summarizes the literature on the nutritional protocols, emphasizing their impacts on bone and muscle biology. In addition, we analyzed several CR studies using Gene Expression Omnibus (GEO) database and identified common transcriptome changes to understand the signaling pathway involved in musculoskeletal tissue. We identified nine novel common genes, out of which five were upregulated (Emc3, Fam134b, Fbxo30, Pip5k1a, and Retsat), and four were downregulated (Gstm2, Per2, Fam78a, and Sel1l3) with CR in muscles. Gene Ontology enrichment analysis revealed that CR regulates several signaling pathways (e.g., circadian gene regulation and rhythm, energy reserve metabolic process, thermogenesis) involved in energy metabolism. In conclusion, this study summarizes the beneficiary role of CR and identifies novel genes and signaling pathways involved in musculoskeletal biology.
... It is confusing to note that decreased IGF-1R combined with increased PTEN shortens the life span of Zmpste24 −/− mice, whereas IGF-1R +/− mice (36) or super-PTEN mice (37) live an extended life span. Previous studies concluded that attenuation of IGF-1/Akt/ mTOR signaling might reflect a defensive response aimed at minimizing cell growth and metabolism in the context of systemic damage (7). ...
... Previous studies concluded that attenuation of IGF-1/Akt/ mTOR signaling might reflect a defensive response aimed at minimizing cell growth and metabolism in the context of systemic damage (7). However, these defensive responses might eventually aggravate health span and accelerate the aging process, even leading to frequent embryonic lethality (29,36,38). Furthermore, we and others (33) have shown that activation of Akt/mTOR signaling is supposed to protect Zmpste24-deficient embryos from development failure possibly induced by genomic instability (6) or defective mitosis (16,17) or to largely rescue Zmpste24-deficient aging phenotypes by targeting the lipid modification of prelamin A (39,40). ...
Progerin, a product of LMNA mutation, leads to multiple nuclear abnormalities in patients with Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disorder. Progerin also accumulates during physiological aging. Here, we demonstrate that impaired insulin-like growth factor 1 receptor (IGF-1R)/Akt signaling pathway results in severe growth retardation and premature aging in Zmpste24 −/− mice, a mouse model of progeria. Mechanistically, progerin mislocalizes outside of the nucleus, interacts with the IGF-1R, and down-regulates its expression, leading to inhibited mitochondrial respiration, retarded cell growth, and accelerated cellular senescence. Pharmacological treatment with the PTEN (phosphatase and tensin homolog deleted on chromosome 10) inhibitor bpV (HOpic) increases Akt activity and improves multiple abnormalities in Zmpste24-deficient mice. These findings provide previously unidentified insights into the role of progerin in regulating the IGF-1R/Akt signaling in HGPS and might be useful for treating LMNA -associated progeroid disorders.
... Mutants with a reduced activity of the insulin/IGF-1-receptor homolog DAF-2 have a double lifespan, and their longevity requires the activity of transcription factor DAF-16, a member of the FOXO (forkhead box transcription factor O) family. The insulin/IGF pathway is also known to regulate longevity and aging in other species, including mammals [58][59][60][61][62]. Lin et al. [63] showed that the DAF-2 acts through a conserved phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. ...
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This review introduces the subject of senescence, aging, and the formation of senescent multinucleated giant cells. We define senescence and aging and describe how molecular and cellular senescence leads to organismal senescence. We review the latest information on senescent cells’ cellular and molecular phenotypes. We describe molecular and cellular features of aging and senescence and the role of multinucleated giant cells in aging-related conditions and cancer. We explain how multinucleated giant cells form and their role in aging arteries and gonads. We also describe how multinucleated giant cells and the reversibility of senescence initiate cancer and lead to cancer progression and metastasis. We also describe molecules and pathways regulating aging and senescence in model systems and their applicability to clinical therapies in age-related diseases.
... No large-scale phenotyping study has been published until recently. For growth hormone signaling, we therefore reviewed all available data obtained from studies involving long-lived dwarf and related mouse lines (Ames dwarf [216], Laron dwarf [217], Snell dwarf [218], growth hormone receptor knock-out [219], growth hormone releasing hormone receptor knock-out [218] and Igf1 heterozygous mice [220]) (Supplementary Table 3). Overall, in all papers analyzed, we identified 61 ASPs examined in both young and old groups of mice (Supplementary Table 3). ...
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Aging is a major risk factor for a number of chronic diseases, including neurodegenerative and cerebrovascular disorders. Aging processes have therefore been discussed as potential targets for the development of novel and broadly effective preventatives or therapeutics for age-related diseases, including those affecting the brain. Mechanisms thought to contribute to aging have been summarized under the term the “hallmarks of aging” and include a loss of proteostasis, mitochondrial dysfunction, altered nutrient sensing, telomere attrition, genomic instability, cellular senescence, stem cell exhaustion, epigenetic alterations and altered intercellular communication. We here examine key claims about the “hallmarks of aging”. Our analysis reveals important weaknesses that preclude strong and definitive conclusions concerning a possible role of these processes in shaping organismal aging rate. Significant ambiguity arises from the overreliance on lifespan as a proxy marker for aging, the use of models with unclear relevance for organismal aging, and the use of study designs that do not allow to properly estimate intervention effects on aging rate. We also discuss future research directions that should be taken to clarify if and to what extent putative aging regulators do in fact interact with aging. These include multidimensional analytical frameworks as well as designs that facilitate the proper assessment of intervention effects on aging rate.
... elegans), have been used to investigate major players in the UPS system and findings have been extrapolated to human cells [38]. A plethora of literature has been reported on the roles of Insulin/IGF, mTOR (mammalian/mechanistic target of rapamycin), the Sirtuins and Forkhead Box O (FOXO) transcription factors in the process of lifespan and longevity (reviewed in [3,[39][40][41][42]). Briefly, reductions in Insulin/IGF and mTOR signaling are observed to modulate lifespan and increase healthspan in various model organisms [43][44][45][46][47][48]. FOXO transcription factors act as key regulators of longevity downstream of insulin/IGF signaling (reviewed in [39,49]). ...
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The development and prevalence of diseases associated with aging presents a global health burden on society. One hallmark of aging is the loss of proteostasis which is caused in part by alterations to the ubiquitin–proteasome system (UPS) and lysosome–autophagy system leading to impaired function and maintenance of mass in tissues such as skeletal muscle. In the instance of skeletal muscle, the impairment of function occurs early in the aging process and is dependent on proteostatic mechanisms. The UPS plays a pivotal role in degradation of misfolded and aggregated proteins. For the purpose of this review, we will discuss the role of the UPS system in the context of age-related loss of muscle mass and function. We highlight the significant role that E3 ubiquitin ligases play in the turnover of key components (e.g., mitochondria and neuromuscular junction) essential to skeletal muscle function and the influence of aging. In addition, we will briefly discuss the contribution of the UPS system to lifespan. By understanding the UPS system as part of the proteostasis network in age-related diseases and disorders such as sarcopenia, new discoveries can be made and new interventions can be developed which will preserve muscle function and maintain quality of life with advancing age.
... The screens for genes that modified dauer formation had been done previously Riddle et al., 1981), identifying daf-2 and age-1 mutants (in previous publications denominated daf-23) as dauer constitutive which means that even in favorable growth conditions, a percentage of those populations would still go into the dauer stage; with mutations in daf-16 suppressing this dauer constitutive phenotype (Vowels and Thomas, 1992). This interplay between AGE-1 and DAF-2, with DAF-16 opposing their functions in lifespan and dauer formation, would culminate in a series of publications identifying these genes as the components of the insulin/IGF-1 signaling (IIS) pathway in the nematode (Morris et al., 1996;Morris et al., 1996;Kimura et al., 1997;Lin et al., 1997;Ogg et al., 1997;Tissenbaum and Ruvkun, 1998), a pathway conserved throughout evolution and that regulates lifespan across different organisms (Tissenbaum and Ruvkun, 1998;Tatar et al., 2001;Barbieri et al., 2003;Blüher et al., 2003;Holzenberger et al., 2003;Hwangbo et al., 2004), including humans (Hwangbo et al., 2004;Suh et al., 2008;Willcox et al., 2008). ...
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Since its introduction as a genetic model organism, Caenorhabditis elegans has yielded insights into the causes of aging. In addition, it has provided a molecular understanding of mechanisms of neurodegeneration, one of the devastating effects of aging. However, C. elegans has been less popular as an animal model to investigate DNA repair and genomic instability, which is a major hallmark of aging and also a cause of many rare neurological disorders. This article provides an overview of DNA repair pathways in C. elegans and the impact of DNA repair on aging hallmarks, such as mitochondrial dysfunction, telomere maintenance, and autophagy. In addition, we discuss how the combination of biological characteristics, new technical tools, and the potential of following precise phenotypic assays through a natural life-course make C. elegans an ideal model organism to study how DNA repair impact neurodegeneration in models of common age-related neurodegenerative diseases.
Modifications of RNA, collectively called the "epitranscriptome", might provide novel biomarkers and innovative targets for interventions in geroscience but are just beginning to be studied in the context of ageing and stress resistance. RNA modifications modulate gene expression by affecting translation initiation and speed, miRNA binding, RNA stability, and RNA degradation. Nonetheless, the precise underlying molecular mechanisms and physiological consequences of most alterations of the epitranscriptome are still only poorly understood. We here systematically review different types of modifications of rRNA, tRNA and mRNA, the methodology to analyze them, current challenges in the field, and human disease associations. Furthermore, we compiled evidence for a connection between individual enzymes, which install RNA modifications, and lifespan in yeast, worm and fly. We also included resistance to different stressors and competitive fitness as search criteria for genes potentially relevant to ageing. Promising candidates identified by this approach include RCM1/NSUN5, RRP8, and F33A8.4/ZCCHC4 that introduce base methylations in rRNA, the methyltransferases DNMT2 and TRM9/ALKBH8, as well as factors involved in the thiolation or A to I editing in tRNA, and finally the m⁶A machinery for mRNA.
The impact of dietary restriction (DR) on disease and aging has received increasing attention in recent years. There is substantial evidence that DR displays many benefits, including reduced inflammation, lowered cardiovascular risk and improved metabolic fitness. In addition, many growing evidence indicates that DR and aging interact through partially overlapping mechanisms in the activation of some conserved nutrient-signaling pathways, mainly the insulin/insulin-like growth factor (IIS) and the mammalian target of Rapamycin (mTOR). Although the involvement of the mTOR pathway and IIS signaling in regulating life span and aging has been studied extensively, the underpinning mechanisms remain elusive. On the other hand, recent discoveries indicate that the aging process can be improve or delay through specific pharmacological approaches. Therefore, this chapter reviews the literature concerning: (i) the emerging insights linking mTOR and IIS signals to various processes related to aging and disease; (ii) recent discoveries on how DR attenuates aging through AMPK and SIRT1 pathways; (iii) discuss the regulatory mechanisms that may delay or improve the aging process from pharmacological discoveries. We also focus on illustrating some potential anti-aging drugs, such as metformin, rapamycin or resveratrol, and verify their actual effects in vivo. In conclusion, systems approaches and polypharmacology to develop anti-aging drugs may be the most effective way to target nutrient-sensing network in improving late-life health.
The increase in human life expectancy over the past 50 years, and with it, in the prevalence of diseases associated with aging, has given new urgency to research related to the mechanisms underlying aging and age-related disorders. Invertebrate models, such as the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, are key tools for understanding the genetic, physiological, and environmental basis of aging. In this chapter, we enumerate how research on these model organisms has provided a detailed view of some of the molecular events involved in normal aging and age-related diseases, and how they can be used to develop antiaging interventions.
Full-text available
Discoveries made in the nematode Caenorhabditis elegans revealed that aging is under genetic control. Since these transformative initial studies, C. elegans has become a premier model system for aging research. Critically, the genes, pathways, and processes that have fundamental roles in organismal aging are deeply conserved throughout evolution. This conservation has led to a wealth of knowledge regarding both the processes that influence aging and the identification of molecular and cellular hallmarks that play a causative role in the physiological decline of organisms. One key feature of age-associated decline is the failure of mechanisms that maintain proper function of the proteome (proteostasis). Here we highlight components of the proteostatic network that act to maintain the proteome and how this network integrates into major longevity signaling pathways. We focus in depth on the heat shock transcription factor 1 (HSF1), the central regulator of gene expression for proteins that maintain the cytosolic and nuclear proteomes, and a key effector of longevity signals.
Full-text available
Mutations in daf-2 and age-1 cause a dramatic increase in longevity as well as developmental arrest at the dauer diapause stage in Caenorhabditis elegans. daf-2 and age-1 encode components of an insulin-like signaling pathway. Both daf-2 and age-1 act at a similar point in the genetic epistasis pathway for dauer arrest and longevity and regulate the activity of the daf-16 gene. Mutations in daf-16 cause a dauer-defective phenotype and are epistatic to the diapause arrest and life span extension phenotypes of daf-2 and age-1 mutants. Here we show that mutations in this pathway also affect fertility and embryonic development. Weak daf-2 alleles, and maternally rescued age-1 alleles that cause life span extension but do not arrest at the dauer stage, also reduce fertility and viability. We find that age-1(hx546) has reduced both maternal and zygotic age-1 activity. daf-16 mutations suppress all of the daf-2 and age-1 phenotypes, including dauer arrest, life span extension, reduced fertility, and viability defects. These data show that insulin signaling, mediated by DAF-2 through the AGE-1 phosphatidylinositol-3-OH kinase, regulates reproduction and embryonic development, as well as dauer diapause and life span, and that DAF-16 transduces these signals. The regulation of fertility, life span, and metabolism by an insulin-like signaling pathway is similar to the endocrine regulation of metabolism and fertility by mammalian insulin signaling.
Full-text available
Gene mutations in invertebrates have been identified that extend life span and enhance resistance to environmental stresses such as ultraviolet light or reactive oxygen species. In mammals, the mechanisms that regulate stress response are poorly understood and no genes are known to increase individual life span. Here we report that targeted mutation of the mouse p66shc gene induces stress resistance and prolongs life span. p66shc is a splice variant of p52shc/p46shc (ref. 2), a cytoplasmic signal transducer involved in the transmission of mitogenic signals from activated receptors to Ras. We show that: (1) p66shc is serine phosphorylated upon treatment with hydrogen peroxide (H2O2) or irradiation with ultraviolet light; (2) ablation of p66shc enhances cellular resistance to apoptosis induced by H2O2 or ultraviolet light; (3) a serine-phosphorylation defective mutant of p66shc cannot restore the normal stress response in p66shc-/- cells; (4) the p53 and p21 stress response is impaired in p66shc-/- cells; (5) p66shc-/- mice have increased resistance to paraquat and a 30% increase in life span. We propose that p66shc is part of a signal transduction pathway that regulates stress apoptotic responses and life span in mammals.
Newborn mice homozygous for a targeted disruption of insulin-like growth factor gene (Igf-1) exhibit a growth deficiency similar in severity to that previously observed in viable Igf-2 null mutants (60% of normal birthweight). Depending on genetic background, some of the Igf-1(-/-) dwarfs die shortly after birth, while others survive and reach adulthood. In contrast, null mutants for the Igf1r gene die invariably at birth of respiratory failure and exhibit a more severe growth deficiency (45% normal size). In addition to generalized organ hypoplasia in Igf1r(-/-) embryos, including the muscles, and developmental delays in ossification, deviations from normalcy were observed in the central nervous system and epidermis. Igf-1(-/-)/Igf1r(-/-) double mutants did not differ in phenotype from Igf1r(-/-) single mutants, while in Igf-2(-)/Igf1r(-/-) and Igf-1(-/-)/Igf-2(-) double mutants, which are phenotypically identical, the dwarfism was further exacerbated (30% normal size). The roles of the IGFs in mouse embryonic development, as revealed from the phenotypic differences between these mutants, are discussed.
Dysfunction of the pancreatic β cell is an important defect in the pathogenesis of type 2 diabetes, although its exact relationship to the insulin resistance is unclear. To determine whether insulin signaling has a functional role in the β cell we have used the Cre-loxP system to specifically inactivate the insulin receptor gene in the β cells. The resultant mice exhibit a selective loss of insulin secretion in response to glucose and a progressive impairment of glucose tolerance. These data indicate an important functional role for the insulin receptor in glucose sensing by the pancreatic β cell and suggest that defects in insulin signaling at the level of the β cell may contribute to the observed alterations in insulin secretion in type 2 diabetes.
Survival patterns and tumor incidence are presented for long lived mouse strains LP, 129, DBA/2, CBA, C57BL/10, C3H, and C3H.K and 8 derived F1 hybrid groups. In almost all strains, males lived longer than females. Overall, the F1 hybrid combinations of noncongenic parents had not only an increased tumor incidence but also an increased lifespan. The lifespan and age specific incidence of cancer observed for the F1 hybrids could not be accurately predicted from the parental characteristics.
Newborn mice homozygous for a targeted disruption of insulin-like growth factor gene (Igf-1) exhibit a growth deficiency similar in severity to that previously observed in viable Igf-2 null mutants (60% of normal birthweight). Depending on genetic background, some of the Igf-1(-/-) dwarfs die shortly after birth, while others survive and reach adulthood. In contrast, null mutants for the Igf1r gene die invariably at birth of respiratory failure and exhibit a more severe growth deficiency (45% normal size). In addition to generalized organ hypoplasia in Igf1r(-/-) embryos, including the muscles, and developmental delays in ossification, deviations from normalcy were observed in the central nervous system and epidermis. Igf-1(-/-)/Igf1r(-/-) double mutants did not differ in phenotype from Igf1r(-/-) single mutants, while in Igf-2(-)/Igf1r(-/-) and Igf-1(-/-)/Igf-2(-) double mutants, which are phenotypically identical, the dwarfism was further exacerbated (30% normal size). The roles of the IGFs in mouse embryonic development, as revealed from the phenotypic differences between these mutants, are discussed.