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

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

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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
(ç http://www.nature.com/nature).
Acknowledgements We thank D. Reinberg for the anti-RPB1 antibody; V. Sartorelli for the p300
expression construct; U. Schibler for Rev-Erb
a
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
interests.
Correspondence and requests for materials should be addressed to S.M.R.
(e-mail: steven.reppert@umassmed.edu).
..............................................................
IGF-1 receptor regulates lifespan and
resistance to oxidative stress in mice
Martin Holzenberger*, Joe
¨
lle Dupont, Bertrand Ducos*,
Patricia Leneuve*, Alain Ge
´
loe
¨
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
ˆ
pital Saint-Antoine, 75571 Paris 12,
France
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
1–3
.
ExamplesincludetherelatedtyrosinekinasereceptorsInR
(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
1/2
mice live on average 26% longer than their wild-type
littermates (P < 0.02). Female Igf1r
1/2
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
1/2
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
1/2
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
4,5
and
insects
6,7
, respectively. Null mutations of the insect insulin-receptor
substrate Chico, which acts downstream from InR, also extends
lifespan
8
. 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
7,8
. 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
9
whereas IGF-1R promotes
growth
10
. 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
3,8
. The phenotypes of long-lived
spontaneous mouse mutants studied so far indicate a probable link
between longevity and growth. The long-lived Prop1
df/df
(Ames
dwarf) and Pit1
dw/dw
(Snell dwarf) mutants
11,12
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
(neo
r
) and two loxP sites (triangles). Exon 3 and neo
r
were then deleted by Cre-lox
recombination, producing the Igf1r
2
(knockout) allele
14,15
. b, Allele-specific RT–PCR
25
revealed that heterozygous Igf1r
þ/2
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
þ/2
animals (bar graph), the relative distributions
(autoradiographic pattern) were unchanged. Receptors were undetectable in Igf1r
2/2
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
þ/2
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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which strongly decreases circulating IGF-I and impairs growth and
development, also increases lifespan
13
. 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
gene
14,15
(Fig. 1a). We found that homozygous null mutants (Igf1r
2/
2
) died at birth, as previously described in a study using classical
insertional mutagenesis
16
. Our Igf1r
þ/2
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
15
,
IGF-1 receptor levels in Igf1r
þ/2
mice were half those in wild type
Igf1r
þ/þ
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
þ/2
males develop
a modest, 8% growth deficit with respect to their Igf1r
þ/þ
litter-
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
15
. 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).
Igf1r
þ/2
mice lived a mean of 26% longer than Igf1r
þ/þ
controls
(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
þ/2
females outlived Igf1r
þ/2
males, whereas the opposite is normally the case in wild-type
populations of the 129/J genetic background
17
(Fig. 2). Thus, in
Igf1r
þ/2
mice, the degree to which lifespan is extended depends on
sex, as has been described for Drosophila mutants with impaired
insulin-like signalling
6,8
. 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
18,19
.
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
19
. We did not
observe accidental deaths, although some of the sporadic mortality
of younger males may have been consequences of fights for
dominance.
Blood parameters (see Methods) were normal. However, serum
IGF-I levels were upregulated in adult Igf1r
þ/2
mice (males,
795 ^ 64 compared with 625 ^ 30 ng ml
21
, P , 0.01; females,
716 ^ 39 compared with 516 ^ 14 ng ml
21
, P , 0.001; n ¼ 8–10
per group) and may reflect an endocrine response to the reduced
availability of IGF-1R
15
. Blood glucose levels in mice that were
deprived of food overnight were unaffected. In fed animals, how-
ever, Igf1r
þ/2
males tended to have higher (þ12%) and Igf1r
þ/2
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
21
; females,
1.58 ^ 0.25 compared with 1.90 ^ 0.25 ng ml
21
; 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
þ/2
males had a
significantly stronger glucose response than controls (P , 0.001).
In Igf1r
þ/2
females, the response was slightly weaker than in wild-
Figure 2 Lifespan extension in Igf1r
þ/2
mice with respect to Igf1r
þ/þ
(WT) mice.
a, Igf1r
þ/2
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
þ/2
mice compared with wild type (P , 0.001,
Cox’s test). b, Igf1r
þ/2
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
þ/2
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
þ/2
males is related to the reportedly com-
promised
b
-cell mass in these mice
20
.
As metabolism may have an important role in ageing, we
explored energy expenditure in Igf1r
þ/2
mice. Body temperature,
indicative of metabolic activity and reported to be low in long-
lived Ames dwarf mice
21
, was unaffected in Igf1r
þ/2
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
þ/2
and Igf1r
þ/þ
mice (data not shown). Further-
more, as caloric restriction is known to extend rodent lifespan, we
investigated the possibility that Igf1r
þ/2
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
þ/2
and Igf1r
þ/þ
mice, with
mean food intake (in g d
21
kg
21
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
21
kg
21
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
21
kg
20.67
; females, 351.9 ^ 6.0 compared with 337.9 ^
7.0 J min
21
kg
20.67
; 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
21
-
kg
20.67
; females, 135.8 ^ 9.4 compared with 140.9 ^ 6.6 J min
21
-
kg
20.67
).
Long-lived C. elegans daf-2 mutants and long-lived dwarf mice
display changes in fertility. We therefore monitored fertility and
reproduction in Igf1r
þ/2
female mice from puberty to the age of 13
months. Igf1r
þ/2
females became fertile at 5.2 ^ 0.1 weeks whereas
Igf1r
þ/þ
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
þ/2
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,
x
2
test).
b, Although offspring decrease markedly with age, there are no consistent differences
between Igf1r
þ/2
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
þ/2
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
þ/2
and absent from Igf1r
2/2
MEFs. b, Anti-IGF-1R
b
,
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
Igf1r
þ/2
and absent from Igf1r
2/2
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
þ/2
and absent from
Igf1r
2/2
MEFs.
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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)
knockout
13
. The litter size of young Igf1r
þ/2
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
þ/2
females and their controls displayed
indistinguishable profiles.
Oxidative stress is a principal cause of ageing
22
, and mouse and fly
mutants with enhanced resistance to oxidative stress are long-
lived
23,24
. We therefore subjected adult mice to oxidative stress by
injection of paraquat, a herbicide that induces formation of reactive
oxygen species. Igf1r
þ/2
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
2
O
2
, and found that the proportion of
surviving cells was significantly higher in Igf1r
þ/2
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
8
and of other
proteins acting downstream of IGF-1R, such as mouse p66 Shc
23
and C. elegans phosphatidylinositol-3-OH kinase (AGE-1)
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
þ/2
and Igf1r
2/2
mice
14,15
and studied signalling by western blot. As expected,
Igf1r
þ/2
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
þ/2
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
þ/2
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
4–6
, also seems to exist in higher vertebrates.
Unlike long-lived mouse mutants with hypopituitarism
11,12,25
or
with complete lack of GHR/BP
13
, long-lived Igf1r
þ/2
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
15
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
26
.Itis
therefore not surprising that we found abnormal regulation of
blood glucose in Igf1r
þ/2
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
14,15
, 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
6,12
. 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
þ/2
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
2/2
is the only other targeted mutation in mammals
described so far that leads to a comparable increase in lifespan
without inducing major side effects
23
. 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
þ/2
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
1
mutants showing that lifespan is extended much
more than can be explained by the modest increase in resistance to
oxidative stress
8
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
unclear
25,27
. Owing to strong oxidative stress-resistance phenotypes
associated with C. elegans longevity mutations
28
, these aspects merit
further study in vertebrates. Our Igf1r
þ/2
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
29
. 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
Methods
Mice
The Igf1r mutant, described elsewhere
14
and available from http://www.emma.rm.cnr.it,
was maintained in the heterozygous state in the 129/Sv genetic background. By mating
Igf1r
þ/2
males with 9- to 12-week-old 129/Sv wild-type females, we generated three
cohorts, each composed of heterozygous Igf1r
þ/2
and Igf1r
þ/þ
(wild type) littermates.
Animals lived in conventional conditions: 23 8C, 14/10-h light/dark cycle, standard diet
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Group
(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
Igf1r
þ/2
and 17 Igf1r
þ/þ
females; 12 Igf1r
þ/2
and 16 Igf1r
þ/þ
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
þ/2
and 15 Igf1r
þ/þ
females; 14 Igf1r
þ/2
and 11 Igf1r
þ/þ
males) was
used for blood biochemistry and analysis of glucose tolerance, food consumption, fertility
and body composition. In cohort 3 (17 Igf1r
þ/2
and 11 Igf1r
þ/þ
females, 14 Igf1r
þ/2
and
15 Igf1r
þ/þ
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
elsewhere
14,15
) and rhdes(1-3)IGF-I (for autoradiography) were labelled with
125
I (see also
Supplementary Information).
For the allele-specific expression assay, we used total RNA from Igf1r
þ/2
embryos and
triplex reverse transcriptase-polymerase chain reaction (RT–PCR)
26
. 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
5
0
-CGCCTGGAAAACTGCACG-3
0
annealed with exon 2, the first reverse primer
5
0
-AGCTGCCCAGGCACTCCG-3
0
annealed with exon 3, and the second reverse primer
5
0
-GCAGGGGATACAGTACATGTTT-3
0
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.
Fertility
We determined the onset of male and female fertility by mating Igf1r
þ/2
and Igf1r
þ/þ
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
offspring.
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
21
body
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
þ/þ
, Igf1r
þ/2
and Igf1r
2/2
embryonic day (E)14
fetuses. IGF signalling pathways and in vitro resistance to H
2
O
2
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
2
O
2
resistance
we treated MEFs with 100
m
MH
2
O
2
and determined cell viability after 1–3 days using
Trypan blue and a haemocytometer. n ¼ 6 for each group, in two independent
experiments.
Western blotting
We performed immunoprecipitation and western blotting as described
30
(see
Supplementary Information for details).
Statistics
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
x
2
tests where
indicated.
Received 4 October; accepted 18 November 2002; doi:10.1038/nature01298.
Published online 4 December 2002.
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letters to nature
NATURE | VOL 421 | 9 JANUARY 2003 | www.nature.com/nature186
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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
interests.
Correspondence and requests for materials should be addressed to M.H.
(e-mail: holzenberger@st-antoine.inserm.fr).
..............................................................
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
1,2
. This response is dependent on the transcription
factor Ste12 and on the mating pheromone-response mitogen-
activated protein (MAP) kinase cascade
1
, 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
limitation
1,2
. This response requires the transcription factors Ste12
and Tec1, which bind cooperatively to filamentous response
elements (FREs) within the promoters of filamentous response
genes
3
. Transcription from FREs is induced in response to nitrogen
starvation
4
, 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
Ste12.
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)
5
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
3
were grown at 30 8C in selective media and assayed for
b
-galactosidase activity
9
. 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
6
. 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
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© 2003
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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.
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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.
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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.