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, The SIRT1 Activator SRT1720 Extends Lifespan and Improves Health of Mice Fed a Standard Diet,
Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.01.031
Cell Reports
Report
MD 21224, USA
2Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards, NSW 2065, Australia
3Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia
4Sirtris, a GSK company, 200 Technology Square, Cambridge, MA 02139, USA
5Glenn Labs for the Biological Mechanisms of Aging, Harvard Medical School, Boston, MA 02115, USA
6School of Public Health, University of Alabama at Birmingham, Birmingham, AL 35294, USA
7Gene Expression and Genomics Unit, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Baltimore,
MD 21224, USA
8These authors contributed equally to this work.
*Correspondence: decabora@grc.nia.nih.gov
http://dx.doi.org/10.1016/j.celrep.2014.01.031
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SRT1720 has been shown to be effective in reducing HFD- inducible transcription factor, Ankyrin repeat domain 1 (Ankrd1),
induced pathologies (Minor et al., 2011), atherosclerosis (Zeng was observed in the muscle following SRT1720 supplementation
et al., 2013), and myocardial infarction (Tong et al., 2013). (Table S3).
Although overexpression of SIRT1 confers a multidrug resis- The effect of SRT1720 on gene expression was further inves-
tance phenotype in various cancer cell lines in vitro (de Jong tigated using parametric analysis of gene set enrichment (PAGE)
et al., 2011; Oh et al., 2010; Zhu et al., 2012), pharmacological (Figure 3C). Interestingly, the negative transcriptional effect of
activation of SIRT1 by SRT1720 has been associated with reduc- SRT1720 was stronger in muscle than in liver. The expanded
tion in multiple myeloma tumor growth (Chauhan et al., 2011), but list of top up- and downregulated pathways in the liver and mus-
increased lung metastasis of breast cancer cells in a mouse cle of SRT1720-treated mice are presented in Tables S4 and S5.
xenograft model (Suzuki et al., 2012). Harmful effects of this Notably, ribosomal proteins were the most upregulated path-
kind warrant further studies on dosage, timing, and actions in ways in liver (Table S4), and alterations in ribosomal biology
various tissues of SRT1720 relevant to the development of ther- and translation are tightly linked to longevity in other organisms
apeutically useful SIRT1 activators. In the study herein, low levels (Heeren et al., 2009; Houtkooper et al., 2013; Rogers et al.,
of serum markers of hepatic and renal functionAST, creatinine, 2011), which may provide another mechanism through which
and CKwere found with SRT1720 treatment (Figure 2C), indi- SRT1720 exerts its prolongevity effects. One of the top path-
cating that the compound was well tolerated. To ensure that ways to be altered by SRT1720 supplementation in both liver
the ingested SRT1720 reached adequate circulating concentra- and muscle was the inflammation pathway, and quantitative
tions, the serum levels of SRT1720 were measured in the morn- RT-PCR analysis showed significant reduction in select proin-
ing and evening and were found to be 193 30.0 and 339 flammatory mediators in both tissues when compared to SD-fed
23.0 ng/ml, respectively, consistent with the feeding cycle of control animals (Figure 3D). Consistent with this idea, circulating
the mice and with our previous study of HFD-fed mice (Minor tumor necrosis factor (TNF)-a levels were lower in the SRT1720
et al., 2011). To date, there has been no correlation between cohort (Figure 3E), and a number of NF-kB target genes were
STAC concentrations (resveratrol or SRT1720) in vivo and acti- among the top downregulated genes following SRT1720 supple-
vation of SIRT1 in vitro, the reason being that there is controversy mentation (Table S3). Similarly, western blotting of liver lysates
surrounding the methods for measuring SIRT1 enzymatic activity illustrated the significant reduction in protein levels encoding
(Dominy et al., 2013). Thus far, the measurement of the steady- for p65Rel and two known targets of NF-kB, COX2, and BAX
state acetylation status of select sirtuin substrates in vivo (e.g., (Figure 3F). No alteration in SIRT1 protein expression was
PGC-1a, p53, NF-kB, and others) remains an important tool for observed. Although the acetylation status of canonical SIRT1
evaluating changes in sirtuin activity (Dominy et al., 2013). Based targets was not directly assessed, these results led us to hypoth-
on our NF-kB data (see below), we propose that the measured esize that SRT1720 may inhibit NF-kB signaling via SIRT1
plasma concentration of SRT1720 in treated mice is sufficient activation.
to activate SIRT1 in vivo. To explore the molecular mechanisms underlying SRT1720
Whole-genome DNA microarrays were performed on liver and control of inflammatory signaling, murine embryonic fibroblasts
muscle samples of SD-fed mice supplemented or not with (MEFs) derived from SIRT1-knockout mice were used for
SRT1720. Principal component analysis showed a distinct sep- cDNA microarray analysis (Figure 4; Table S6) in conjunction
aration of both treatment groups, with the effect of SRT1720 with phosphoantibody microarray (Table S7). Using the latter
supplementation being more pronounced in the muscle tissue method, it has previously been demonstrated that SIRT1 dele-
(Figure 3A versus 3B). The top ten up- and downregulated genes tion is associated with constitutive activation of the proinflamma-
in both tissues are shown in Table S3, with the complete data set tory NF-kB pathway (Bernier et al., 2011). Overexpression of
made available at http://ncbi.nlm.nih.gov/geo/. Among the top SIRT1 is associated with reduced NF-kB activity in mice (Pfluger
upregulated genes, members of the cytokine-induced STAT in- et al., 2008), whereas SIRT1 activation by SRT1720 reduces
hibitor (CIS) family (e.g., suppressor of cytokine signaling 2 expression of NF-kB and its targets in foam cells (Zeng et al.,
[SOCS2] and cytokine-inducible SH2-containing protein 2013) and multiple myeloma cells (Chauhan et al., 2011). Here,
[CISH]), which are associated with suppression of the inflamma- it was found that the expression level of a few genes was influ-
tory response (Kubo et al., 2003), were significantly higher in the enced by SRT1720 in a SIRT1-independent manner (e.g., Id2),
liver and muscle of SRT1720-treated mice. Similarly, a 7-fold in- whereas the expression of other genes, including Has2,
crease in hepatic expression of cytochrome P450 1A2, which Gadd45a, Ddit3, Thbs1, and Ifitm3, required SIRT1 following
has a role in xenobiotic, cholesterol, and lipid metabolism (Haf- MEF stimulation with SRT1720 (Table S6). Importantly,
ner et al., 2011), was observed in SRT1720-treated mice and SRT1720 induced significant changes in the phosphorylation
may explain the lower circulating serum cholesterol and LDL status of several proteins involved in NF-kB activation in wild-
levels in these animals. These results are consistent with a recent type MEFs, but not Sirt1-KO MEFs (Table S7). Given that low-
report showing that SRT1720 ameliorates fatty liver through a grade chronic inflammation is believed to contribute to aging
reduction in lipogenic enzyme expression in monosodium gluta- and age-related diseases (Franceschi et al., 2000), our results
mate-treated mice (Yamazaki et al., 2009). Lipocalin 2 (Lcn2) and indicate that SRT1720 may keep nonobese mice healthier and
serum amyloid A1 and A2 (Saa1, Saa2), which are involved in extend their mean lifespan by spurring robust inflammatory de-
innate immunity and the acute phase response, respectively, fense. Indeed, SRT1720 exerts antiinflammatory properties in
were the top three most downregulated genes in the liver of mouse models of asthma (Ichikawa et al., 2013) and emphysema
SRT1720-treated animals. Downregulation of the cytokine- (Yao et al., 2012).
Figure 3. SRT1720 Elicits Differential Gene Expression Profiles in the Liver and Muscle of SD-Fed Mice
(A and B) Principal component analysis (PCA) was performed on liver (A) and skeletal (B) muscle of SD-fed mice supplemented without and with SRT1720.
(C) Parametric analysis of gene-set enrichment (PAGE) analysis was performed on microarray data. Columns show significantly up- (red) and downregulated
(blue) pathways following SRT1720 supplementation.
(D) mRNA expression analysis in liver and skeletal muscle by quantitative real-time PCR. Relative expression values were normalized to those of SD-fed control mice.
(E) Serum tumor necrosis factor alpha (TNF-a) concentrations in SD-fed mice supplemented or not with SRT1720. Data are shown as mean SEM.
(F) Western blotting of liver lysates from SD-fed mice supplemented or not with SRT1720. Upper-left panel, representative blots; remaining panels, Signals
associated with bands of interest were normalized to GAPDH and plotted.
*p % 0.05; **p % 0.01; ***p % 0.001 in comparison to SD-fed control animals.
In summary, we report data showing that SRT1720 elicits life- (drug/kg body weight). Study diets were purchased from Dyets. SRT1720
span extension and improved healthspan in mice maintained was provided by Sirtris Pharmaceuticals. All groups had ad libitum access
to their prescribed diet and water throughout the study. Body weight and
on a standard diet. This work highlights the importance of exam-
food intake were monitored biweekly. Animal rooms were maintained at 20
ining the therapeutic value of small molecule activators of SIRT1 22 C with 30%70% relative humidity and a 12 hr light/dark cycle. All animal
in general health and longevity. protocols were approved by the Animal Care and Use Committee (352-LEG-
2012) of the National Institute on Aging.
EXPERIMENTAL PROCEDURES
Survival Study
Animals and Diets Moribund animals were euthanized, and every animal found dead or eutha-
Male C57BL/6J mice were obtained from the Jackson Laboratory at 15 weeks nized was necropsied. Additional information is available in the Supplemental
of age and housed at the Gerontology Research Center, in Baltimore, MD, in Information.
cages of four. Diets were started at 28 weeks of age after randomization into
four groups of 100 mice per group. Mice were fed one of four diets: standard Histology
diet (SD) of AIN-93G (carbohydrate:protein:fat ratio of 64:19:17 percent of Organs were fixed in 4% paraformaldehyde then stained with hematoxylin and
kcal), an SD supplemented with SRT1720 (SD-SRT1720), a high-fat diet eosin for scoring of pathology by a histopathologist blinded to the treatment
consisting of AIN-93G modified to provide 60% of calories from fat (HFD; group.
carbohydrate:protein:fat ratio of 16:23:61), or a HFD supplemented with
SRT1720 (HFD-SRT1720). SRT1720 was included in diets at a concentration Determination of Serum Marker Concentrations and HOMA-IR
of 1.33 g/kg (SD) and 2 g/kg (HFD; g drug/kg chow), respectively, which was Calculation
formulated to provide mice with approximate daily doses of 100 mg/kg Detailed information can be found in the Supplemental Information.
Phosphoprotein Profiling
The Phospho Explorer antibody microarray from Full Moon Biosystems was
used to survey the phosphorylation state of select proteins involved in the
IKK/NF-kB activation pathway, according to the protocol outlined in Bernier
et al. (2011). In brief, wild-type and Sirt1-KO murine embryonic fibroblasts
(gift of Raul Mostoslavsky, Harvard Medical School) were incubated with
vehicle or 3 mM SRT1720 for 18 hr, after which cell lysates were prepared
and biotinylated. The antibody array experiment was performed by Full
Moon Biosystems and the data were analyzed with GenePix Pro 6.0 (Molecular
Devices). Each of the 1,318 antibodies had two replicates printed on a coated
microscope slide, along with multiple positive and negative controls.
Statistics
Figure 4. SIRT1 Dependence in SRT1720-Mediated Gene Expres-
Data are expressed as means SEM. Students t tests were used for all
sion in MEF Cells
comparisons unless otherwise stated. Mortality during the survival study
Microarray data collected from untreated (UT) and SRT1720-treated wild-type
was assessed through the use of the nonparametric log-rank (Mantel-
(WT) and Sirt1-KO MEFs were used to illustrate enrichment of select genes. A
Haenszel) test to compare the differences in Kaplan-Meier survival curves.
larger list of genes can be found in Table S6.
Median survival was assessed using the nonparametric chi-square test (Su
and Wei, 1993). Ninetieth percentile survival estimates were used as surrogate
Oral Glucose Tolerance Test measures of maximum survival times according to the methods of Wang et al.
Following an overnight fast (18 hr), mice received an oral gavage of 30% (2004). The survival analyses were implemented in R (The R Development Core
glucose solution (Sigma-Aldrich) at a dose of 2 g/kg body weight. Blood Team) from scratch using the methodological references given. Comparisons
glucose was measured using an Ascensia Elite glucose meter at 0, 15, 30, for the histopathology data were performed using Fishers exact test. Analyses
60, and 120 min following gavage (n = 6 SD, n = 8 SD-SRT1720, 73 weeks were performed using Excel 2010 (Microsoft), IBM SPSS Statistics, or
age, 46 weeks on diet). SigmaStat 3.0 (Aspire Software International). A p value of % 0.05 was consid-
ered statistically significant.
Body Composition
Measurements of lean body mass, fat, and fluid mass in live mice were
ACCESSION NUMBERS
acquired by nuclear magnetic resonance spectroscopy using the Minispec
LF90 (Bruker Optics) (n = 84 SD, n = 93 SD-SRT1720, n = 51 HFD, n = 73
All raw data were deposited to the NCBI Gene Expression Omnibus under
HFD-SRT1720, 76 weeks age, 49 weeks on diet).
accession number GSE50987.
Metabolic Assessment
Mouse metabolic rate was assessed by indirect calorimetry in open-circuit SUPPLEMENTAL INFORMATION
Oxymax chambers with CLAMS (Columbus Instruments) as previously
described (Minor et al., 2011) (n = 8 SD, n = 8 SD-SRT1720, n = 7 HFD, Supplemental Information includes Supplemental Experimental Procedures,
n = 8 HFD-SRT1720, 52 weeks of age, 25 weeks on diet). two figures, and seven tables and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2014.01.031.
Rotarod
All mice were acclimated 15 min before testing. Mice were tested at the same ACKNOWLEDGMENTS
time of day during their light cycle over a 5-day period. Mice were given a habit-
uation trial at a constant speed of 4 rpm for 1 min before the first trial. On the This research was conducted under a Cooperative Research and Develop-
same day, there was a total of three trials given, separated by 30 min rest ment Agreement (CRADA) between Sirtris, a GSK Company, and the National
periods, during which the rotarod accelerated from 4 to 40 rpm over a period Institute on Aging, National Institutes of Health (NIA/NIH). Funding was pro-
of 5 min. Latency to fall was recorded and averaged over all three trials. This vided by the Intramural Research Program of the NIA/NIH. We are grateful
test was completed on the same mice at 13, 18, and 24 months of age. to Dawn Nines, Justine Lucas, and Dawn Phillips-Boyer for their excellent an-
imal care. We thank Dr. Elin Lehrmann for microarray assistance. We thank
Cataract Formation Drs. Hua-Jun He (National Institute of Standards and Technology, Gaithers-
Lens opacity reading was performed using a Kowa SL14 hand-held slit lamp burg, MD), Yaping Zong (Full Moon Biosystems, Inc., Sunnyvale, CA), and
(Kowa) by an experienced pathologist blinded to the experimental group, Xiong Li (Department of Mathematics and Computer Science, Emory Univer-
as described previously (Wolf et al., 2005) (n = 138 eyes SD, n = 142 eyes sity, Atlanta, GA) for their contribution in carrying out the phosphoantibody
SD-SRT1720, 105 weeks age, 78 weeks diet). array experiment and data analysis. We thank Dr. Norm Wolf for the assess-
ment of cataracts in the mice and Olga Carlson for technical assistance with
Western Blotting the multiplex assay. S.J.M. was supported by a National Medical Health and
Detailed information can be found in the Supplemental Information. Research Council of Australia CJ Martin Early Career Fellowship (RGMS ID
2010-01671). J.D. is supported by a University of Alabama at Birmingham Sta-
Microarray tistical Genetics Postdoctoral Training Program Grant (T32HL072757). D.A.S.
RNA from tissues was isolated using the RNeasy kit (QIAGEN) and then consults for and G.P.V. and J.L.E. are employed by Sirtris, a GSK Company
hybridized to BD-202-0202 Illumina BeadChips. Raw data were subjected to that has a commercial interest in developing SIRT1 activators.
Received: July 18, 2013 Houtkooper, R.H., Mouchiroud, L., Ryu, D., Moullan, N., Katsyuba, E., Knott,
Revised: December 16, 2013 G., Williams, R.W., and Auwerx, J. (2013). Mitonuclear protein imbalance as
Accepted: January 23, 2014 a conserved longevity mechanism. Nature 497, 451457.
Published: February 27, 2014 Hubbard, B.P., Gomes, A.P., Dai, H., Li, J., Case, A.W., Considine, T., Riera,
T.V., Lee, J.E., e, S.Y., Lamming, D.W., et al. (2013). Evidence for a common
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