Report

Ribosomal Protein S6 Kinase 1 Signaling Regulates Mammalian Life Span

See allHide authors and affiliations

Science  02 Oct 2009:
Vol. 326, Issue 5949, pp. 140-144
DOI: 10.1126/science.1177221

This article has a correction. Please see:

Mimicking Caloric Restriction

The extended life span and resistance to age-related diseases in animals exposed to caloric restriction has focused attention on the biochemical mechanisms that produce these effects. Selman et al. (p. 140; see the Perspective by Kaeberlein and Kapahi) explored the role of the mammalian ribosomal protein S6 kinase 1 (S6K1), which regulates protein translation and cellular energy metabolism. Female knockout mice lacking expression of S6K1 showed characteristics of animals exposed to caloric restriction, including improved health and increased longevity. The beneficial effects included reduced fat mass in spite of increased food intake. Thus, inhibition of signaling pathways activated by S6K1 might prove beneficial in protecting against age-related disease.

Abstract

Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)–activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.

Genetic studies in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster implicate several mechanisms in the regulation of life span. These include the insulin and insulin-like growth factor 1 (IGF-1) signaling (IIS) pathway and the mammalian target of rapamycin (mTOR) pathway, which both activate the downstream effector ribosomal protein S6 kinase 1 (S6K1) (1, 2). Although the role of these pathways in mammalian aging is less clear, there is mounting evidence that IIS regulates life span in mice (1). Global deletion of one allele of the IGF-1 receptor (Igf1r), adipose-specific deletion of the insulin receptor (Insr), global deletion of insulin receptor substrate protein 1 (Irs1), or neuron-specific deletion of Irs2, all increase mouse life span (1). Life-span–extending mutations in the somatotropic axis also appear to work through attenuated IIS (3). Igf1r has also been implicated as a modulator of human longevity (4). However, the action of downstream effectors of IIS or mTOR signaling in mammalian longevity is not fully understood.

S6K1 transduces anabolic signals that indicate nutritional status to regulate cell size and growth and metabolism through various mechanisms (5). These include effects on the translational machinery and on cellular energy levels through the activity of adenosine monophosphate (AMP)–activated protein kinase (AMPK) (6, 7). Furthermore, S6K1 serine phosphorylates IRS1 and IRS2, which decreases insulin signaling (5). Given the key role of S6K1 in IIS and mTOR signaling and the regulation of aging in lower organisms by mTOR, S6K, and their downstream effectors (2), we used log-rank testing to evaluate differences in life span of wild-type (WT) and S6K1−/− littermate mice on a C57BL/6 background (8, 9). Data for both sexes combined showed median life-span in S6K1−/− mice increased by 80 days (from 862 to 942 days) or 9% relative to that of WT mice (χ2 = 10.52, P < 0.001) (Fig. 1A and Table 1). Maximum life span (mean life span of the oldest 10% within a cohort) was also increased (1077 ± 16 and 1175 ± 24 days, P < 0.01 for WT and S6K1−/− mice, respectively). Analysis of each sex separately showed that median life span in female S6K1−/− mice was increased by 153 days (from 829 to 982 days) or 19% relative to that of WT mice (χ2 = 11.07, P < 0.001) (Fig. 1B and Table 1). Female maximum life-span was also increased (Table 1). In contrast, deletion of S6K1 in male mice had no effect on median (χ2 = 0.34, P > 0.05) (Fig. 1C and Table 1) or maximum life span (Table 1). Similar gender effects on life span have been reported in other long-lived IIS mouse mutants (8, 10). Cox regression analysis of pooled male and female life-span data revealed no effect of recruitment date, parental identity, or gender, but that of genotype was significant (table S1). Therefore, deletion of S6K1 increases longevity in female mice.

Fig. 1

Extended life span of mice with deletion of S6K1−/−. (A) Kaplan-Meier survival curves for combined male and female wild-type (WT) and S6K1−/− mice show a significant (log-rank χ2 = 10.52, P < 0.001) life-span extension in S6K1−/− mice (n = 49 for WT mice and n = 48 for S6K1−/− mice). (B) Life-span extension was observed in female S6K1−/− mice (χ2 = 11.07, P < 0.001; n = 23 for WT mice and n = 29 for S6K1−/− mice). (C) No significant increase in life span in S6K1−/− mice (χ2 = 0.34, P > 0.05; n = 26 for WT mice and n = 19 for S6K1−/− mice).

Table 1

Comparative survival characteristics of S6K1−/− and WT mice. Oldest (youngest) 10% are the mean life span of the longest (or shortest) living 10% of animals within a genotype. Values are reported ±SEM, where appropriate.

View this table:

Female S6K1−/− mice also showed improvements in a number of age-sensitive biomarkers of aging. In forced motor activity on a rotating rod (rotarod) assays to assess motor and neurological function, 600-day-old female S6K1−/− mice performed better than WT littermates (Fig. 2A). Performance in open-field testing to analyze general activity and exploratory drive were also enhanced (Fig. 2, B and C). An increase in abundance of memory T cells and a reduced number of naïve T cells are seen in mice with age, and the extent of these changes may be correlated with longevity (11). Female S6K1−/− mice at 600 days of age had significantly fewer memory and more naïve T cells than did WT mice (Fig. 2D), although male mice also displayed this phenotype (fig. S1A). Micro– computed tomography scanning of tibia from 600-day-old female S6K1−/− mice revealed attenuation of the normal age-dependent loss of cancellous bone volume seen in C57BL/6 mice (12) (Fig. 2, E and F). However, there was no difference in the incidence of macroscopic tumors in S6K1−/− and WT animals [8% (4 out of 48) for S6K1−/− and 8% (4 out of 49) for WT mice, respectively.

Fig. 2

Age-related pathology and physiological characteristics of 600-day-old female S6K1−/− mice. (A) S6K1−/− mice had improved rotarod performance. (B and C) Increased general activity and exploratory drive was observed in S6K1−/− mice. (D) Abundance of memory and naïve T cells in WT and S6K1−/− mice. (E and F) Bone volume and trabecular number in WT and S6K1−/− mice. (G and H) Insulin sensitivity and glucose tolerance of WT and S6K1−/− mice. (I) S6K1−/− mice were lean and (J) had reduced plasma leptin levels. (K) Body mass (P < 0.01 at all time points), total circulating IGF-1 (L), and pituitary growth hormone (GH) concentrations in WT and S6K1−/− mice (M). Values are means ±SEM. Asterisks indicate statistical difference compared with WT mice by using two-tailed t tests, *P < 0.05; **P < 0.01; and ***P < 0.001; n = 6 to 8 per genotype.

Young, male S6K1−/− mice fed a high-fat diet (13) display increased insulin sensitivity and reduced adiposity relative to those of WT mice, phenotypes also seen in WT mice under caloric restriction (CR), an evolutionarily conserved environmental manipulation that extends life span (14). Insulin sensitivity (assessed by the updated homeostasis model, HOMA2) was significantly greater in 600-day-old female S6K1−/− mice than in WT animals (Fig. 2G), and glucose tolerance was improved (Fig. 2H), in contrast to the impaired glucose tolerance seen in young animals (fig S1B). Fat mass and plasma leptin levels were lower in old female S6K1−/− mice (Fig. 2, I and J), despite increased food intake (fig. S1C). Core temperature and resting metabolic rate (with general linear modeling to account for body-mass differences) were not significantly different (fig. S1, D and E). Although S6K1−/− mice were smaller than their littermates throughout their lives (Fig. 2K), endocrinologically they did not resemble long-lived pituitary dwarfs (15), because their total circulating IGF-1, pituitary growth hormone, thyroid-stimulating hormone, and prolactin concentrations were normal (Fig. 2, L and M, and fig. S1, F and G). Male S6K1−/− mice at 600 days of age had normal fasting and fed glucose levels (fig. S1, H and I).

We compared the effect of S6K1 deletion on genome-wide hepatic gene expression in 600-day-old female mice to transcriptional changes induced by long-term CR (16). The 500 gene categories most overrepresented among genes with altered expression in S6K1−/− mice showed a highly significant overlap with categories overrepresented among CR-regulated genes (P = 3.25 × 10−42 and P = 1.15 × 10−19 for up- and down-regulated categories, respectively, Fisher’s exact test) (fig. S2A). Hepatic transcript profiles in long-lived Irs1−/− mice (8) were also similar (P = 1.60 × 10−21 and P = 8.61 × 10−20 for up- and down-regulated categories, respectively) (fig. S2B). Furthermore, we observed significant correlations in the directions of transcriptional changes associated with highly significant functional categories (P < 10−4, two-tailed) in both comparisons (fig. S2, A and B). This is consistent with the existence of common mechanisms underlying the effects of S6K1, CR, and IIS on aging.

We examined transcription of individual genes in liver, skeletal muscle, and white adipose tissue (WAT) in 600-day-old female S6K1−/− and WT mice, looking for genes previously associated with longevity (tables S2, A and B, S3, A and B, and S4, A and B). Significant cross talk exists between peroxisome proliferator–activated receptor (PPAR)–γ, coactivator 1 α (PGC-1α), AMPK, and nicotinamide adenine dinucleotide (oxidized form)–dependent deacetylase sirtuin-1 (SIRT1) signaling, which may be critical to cellular energy metabolism and perhaps aging (17). Increased expression of genes associated with these pathways was observed in liver (Ppargc1a, Ppargc1b, Foxo1, Foxo3a, Cpt1b, Pdk4, Glut1, and Cyc) and muscle (Ppargc1a, Ppara, Foxo1, Foxo3a, Pdk4, Glut1, Sirt1, and Ucp3) of S6K1−/− mice. Adipose tissue is a key tissue in longevity assurance in C. elegans, D. melanogaster, and mice (18). In WAT of S6K1−/− mice, fewer PGC-1α–regulated genes (Foxo3a, Pdk4, Nampt, and Angptl4) showed increased expression compared with changes seen in liver and muscle, but there was also increased expression of the α2 catalytic and β1 regulatory subunits of AMPK (log 2 fold change = 1.7, P = 2.88 × 10−6 and 1.2, P = 4.95 × 10−5, respectively, Cyber-T analysis). AMPK activity is increased in WAT, muscle, and liver of S6K1−/− mice (7). Moreover, comparison of gene expression patterns in muscle of S6K1−/− mice with those of mice treated with the AMPK activator aminoimidazole carboxamide ribonucleotide (AICAR) (19) revealed a strong overlap between gene categories that showed increased expression, including those associated with PPAR signaling and lipid metabolism (fig. S2C). We confirmed enhanced AMPK activation by AICAR in isolated hepatocytes from S6K1−/−mice (Fig. 3A).

Fig. 3

Enhanced AMPK activation by AICAR of S6K1−/− hepatocytes, increased AAK-2 phosphorylation in rsks-1(ok1255) mutants, and effects of loss of aak-2(ok524) on longevity and physiology. (A) AICAR-stimulated AMPKα2 activity in isolated hepatocytes from S6K1−/− mice. (B) Phosphorylation of AAK-2 Thr243 in rsks-1(ok1255) null mutants subjected, or not, to RNA interference for par-4, the worm LKB kinase that effects this phosphorylation. (C) Life span of rsks-1(ok1255) nulls with mutation of aak-2(ok524). (D to F) Body length, onset of reproductive function, and brood-size phenotypes in rsks-1(ok1255) mutants with or without aak-2(ok524) mutation. In (D), rsks-1(ok1255) is significantly different (P < 0.001; one-way ANOVA) from all other groups from day 2 onward, but rsks-1(ok1255);aak-2 is not significantly different from WT or aak-2. (A) to (C) show data from one representative experiment, and (D to F) show combined data from three similar independent experiments. Values (A and D to F) are means ±SEM. In (A), n = 3, and in (D), n > 8 for each strain and time point. For (E) and (F), n > 20 for each group. Asterisks indicate statistical differences by using two-tailed t tests, *P < 0.05, ***P < 0.001.

In C. elegans, AMPK mediates the effects on life-span of one particular form of CR (20) and perturbing IIS (21, 22), which raises the possibility that the longevity of S6K1−/− mice results from increased AMPK activity. To test this, we studied long-lived C. elegans rsks-1(ok1255) null mutants, which lack the single worm S6K1 homolog. rsks-1 mutants showed increased phosphorylation of the worm AMPK catalytic subunit AAK-2 (Fig. 3B), consistent with increased AMPK activity. These findings imply that in worms, as in mice, loss of S6K1 increases AMPK activity. rsks-1 mutants are long-lived (Fig. 3C, and table S5), with reduced and delayed fecundity and, like S6K1−/− mice, reduced body size (Fig. 3, D to F, and table S5), characteristics that could be attributed to reduced nutrient availability or possibly reduced overall translation (23). To test the role of AMPK in mediating the effects of rsks-1 on longevity, we generated mutants lacking both rsks-1 and aak-2. The aak-2(ok524) null allele fully suppressed rsks-1 mutant longevity (Fig. 3C, and table S5). This effect is likely to be specific, because several other modes of C. elegans longevity are not aak-2–dependent (22). Moreover, aak-2(ok524) also suppressed the fecundity and body size defects of rsks-1 mutants (Fig. 3, D to F). This also suggests that these defects do not reflect reduced overall translation; in fact, in muscle cells from growth-deficient S6K1-null mice, protein synthesis is reportedly not reduced (24). Taken together, these results imply that increased AMPK activity may contribute to the longevity of both C. elegans and mice lacking S6K1.

Our studies indicate that S6K1 signaling influences mammalian life span and age-related pathology. S6K1 is regulated in response to nutrient and hormonal signals and may thus participate in the response to CR. mTOR and AMPK are amenable to pharmacological intervention (25, 26). It might be possible to develop drug treatments that manipulate S6K1 and AMPK to achieve improved overall health in later life. Indeed, short-term rapamycin treatment reduces adiposity in mice (27), and metformin treatment extends life span in short-lived mice (28). Furthermore, recently it has been demonstrated that rapamycin treatment initiated late in life extends life span in mice (29). Our results suggest that this may occur via inhibition of S6K1, and together, these studies indicate the feasibility of manipulating mTOR/S6K1 signaling in the treatment of aging-related disease.

Supporting Online Material

www.sciencemag.org/cgi/content/full/326/5949/140/DC1

Materials and Methods

Figs. S1 and S2

Tables S1 to S5

References

  • * Present address: Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK.

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Supported by a Wellcome Trust Functional Genomics award to J.M.T., L.P., D.G., and D.J.W.; a Wellcome Trust Strategic Award to J.M.T., L.P., D.G., and D.J.W.; and grants from the Medical Research Council, Research into Aging, and the Biological and Biotechnology Research Council to D.J.W.
View Abstract

Navigate This Article