Gladstone scientists discover novel mechanism by which calorie restriction influences longevity

10/8/13 1:24 PM

Public release date: 6-Dec-2012

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Contact: Anne Holden
anne.holden@gladstone.ucsf.edu
415-734-2534
Gladstone Institutes

Gladstone scientists discover novel mechanism by which

calorie restriction influences longevity
Breakthrough suggests way to protect cells from damage caused by chronic disease
SAN FRANCISCO, CADecember 6, 2012Scientists at the
Gladstone Institutes have identified a novel mechanism by which
a type of low-carb, low-calorie dietcalled a "ketogenic diet"
could delay the effects of aging. This fundamental discovery
reveals how such a diet could slow the aging process and may
one day allow scientists to better treat or prevent age-related
diseases, including heart disease, Alzheimer's disease and many
forms of cancer.
As the aging population continues to grow, age-related illnesses
have become increasingly common. Already in the United States,
nearly one in six people are over the age of 65. Heart disease
continues to be the nation's number one killer, with cancer and
Alzheimer's close behind. Such diseases place tremendous strain
on patients, families and our healthcare system. But today,
researchers in the laboratory of Gladstone Senior Investigator
Eric Verdin, MD, have identified the role that a chemical
compound in the human body plays in the aging processand
which may be key to new therapies for treating or preventing a
variety of age-related diseases.
In the latest issue of the journal Science, available online today,
IMAGE: Dr. Verdin's team has
Dr. Verdin and his team examined the role of the compound uncovered the specific mechanism by
hydroxybutyrate (OHB), a so-called "ketone body" that is
which a low-calorie, "ketogenic " diet
produced during a prolonged low-calorie or ketogenic diet. While delays the aging process.
ketone bodies such as OHB can be toxic when present at very
Click here for more information.
high concentrations in people with diseases such as Type I
diabetes, Dr. Verdin and colleagues found that at lower
concentrations, OHB helps protect cells from "oxidative stress"which occurs as certain molecules
build to toxic levels in the body and contributes to the aging process.
"Over the years, studies have found that restricting calories slows aging and increases longevity
however the mechanism of this effect has remained elusive" Dr. Verdin said. Dr. Verdin, the paper's
senior author, directs the Center for HIV & Aging at Gladstone and is also a professor at the
University of California, San Francisco, with which Gladstone is affiliated. "Here, we find that OHB
the body's major source of energy during exercise or fastingblocks a class of enzymes that would
otherwise promote oxidative stress, thus protecting cells from aging."
Oxidative stress occurs as cells use oxygen to produce energy, but this activity also releases other
potentially toxic molecules, known as free radicals. As cells age, they become less effective in clearing
these free radicalsleading to cell damage, oxidative stress and the effects of aging.
However, Dr. Verdin and his team found that OHB might actually help delay this process. In a series
of laboratory experimentsfirst in human cells in a dish and then in tissues taken from micethe
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Gladstone scientists discover novel mechanism by which calorie restriction influences longevity

10/8/13 1:24 PM

team monitored the biochemical changes that occur when OHB is administered during a chronic
calorie-restricted diet. The researchers found that calorie restriction spurs OHB production, which
blocked the activity of a class of enzymes called histone deacetylases, or HDACs.
Normally HDACs keep a pair of genes, called Foxo3a and Mt2, switched off. But increased levels of
OHB block the HDACs from doing so, which by default activates the two genes. Once activated,
these genes kick-start a process that helps cells resist oxidative stress. This discovery not only
identifies a novel signaling role for OHB, but it could also represent a way to slow the detrimental
effects of aging in all cells of the body.
"This breakthrough also greatly advances our understanding of the underlying mechanism behind
HDACs, which had already been known to be involved in aging and neurological disease," said
Gladstone Investigator Katerina Akassoglou, PhD, an expert in neurological diseases and one of the
paper's co-authors. "The findings could be relevant for a wide range of neurological conditions, such
as Alzheimer's, Parkinson's, autism and traumatic brain injurydiseases that afflict millions and for
which there are few treatment options."
"Identifying OHB as a link between caloric restriction and protection from oxidative stress opens up
a variety of new avenues to researchers for combating disease," said Tadahiro Shimazu, a Gladstone
postdoctoral fellow and the paper's lead author. "In the future, we will continue to explore the role of
OHBespecially how it affects the body's other organs, such as the heart or brainto confirm
whether the compound's protective effects can be applied throughout the body."
###
Matthew Hirschey, PhD; John Newman, MD, PhD; Wenjuan He, PhD; Kotaro Shirakawa, PhD; Natacha
Le Moan, PhD; Carrie Grueter, PhD; Hyungwook Lim, PhD; Laura Saunders, PhD; Robert V. Farese,
Jr., MD; and Katerina Akassoglou, PhD, also participated in this research at Gladstone. This research
was supported by the Gladstone Institutes.
About the Gladstone Institutes
Gladstone is an independent and nonprofit biomedical-research organization dedicated to accelerating
the pace of scientific discovery and innovation to prevent, treat and cure cardiovascular, viral and
neurological diseases. Gladstone is affiliated with the University of California, San Francisco.

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References and Notes

1. D. Schluter, The Ecology of Adaptive Radiation (Oxford
Univ. Press, Cambridge, 2000).
2. G. G. Simpson, Tempo and Mode in Evolution (Columbia
Univ. Press, New York, 1944).
3. R. Lande, Evolution 33, 402 (1979).
4. E. I. Svensson, R. Calsbeek, The Adaptive Landscape in
Evolutionary Biology (Oxford Univ. Press, Oxford, 2012).
5. S. Wright, Proc. VI Int. Cong. Gen. 1, 356 (1932).
6. M. Foote, Annu. Rev. Ecol. Syst. 28, 129 (1997).
7. D. L. Rabosky, I. J. Lovette, Proc. Biol. Sci. 275, 2363
(2008).
8. L. J. Harmon et al., Evolution 64, 2385 (2010).

9. J. G. Kingsolver et al., Am. Nat. 157, 245 (2001).

10. C. H. Martin, Am. Nat. 180, E90 (2012).
11. A. P. Hendry, S. K. Huber, L. F. De Len, A. Herrel,
J. Podos, Proc. Biol. Sci. 276, 753 (2009).
12. D. Schluter, Science 266, 798 (1994).
13. C. W. Benkman, Evolution 57, 1176 (2003).
14. R. Calsbeek, D. J. Irschick, Evolution 61, 2493 (2007).
15. C. S. McBride, M. C. Singer, PLoS Biol. 8, e1000529
(2010).
16. E. Svensson, B. Sinervo, Evolution 54, 1396 (2000).
17. D. W. Schemske, H. D. Bradshaw Jr., Proc. Natl. Acad.
Sci. U.S.A. 96, 11910 (1999).
18. D. Schluter, P. R. Grant, Am. Nat. 123, 175 (1984).
19. C. H. Martin, P. C. Wainwright, Evolution 65, 2197 (2011).
20. Additional supporting data and analyses are available in
the supplementary materials on Science Online.
Acknowledgments: Funded by NSF grant DDIG DEB-1010849,
the Gerace Research Centre, and the Center for Population

Suppression of Oxidative Stress by

b-Hydroxybutyrate, an Endogenous
Histone Deacetylase Inhibitor
Tadahiro Shimazu,1,2 Matthew D. Hirschey,1,2 John Newman,1,2 Wenjuan He,1,2
Kotaro Shirakawa,1,2 Natacha Le Moan,3 Carrie A. Grueter,4,5 Hyungwook Lim,1,2
Laura R. Saunders,1,2 Robert D. Stevens,6 Christopher B. Newgard,6 Robert V. Farese Jr.,2,4,5
Rafael de Cabo,7 Scott Ulrich,8 Katerina Akassoglou,3 Eric Verdin1,2*
Concentrations of acetylcoenzyme A and nicotinamide adenine dinucleotide (NAD+) affect
histone acetylation and thereby couple cellular metabolic status and transcriptional regulation.
We report that the ketone body D-b-hydroxybutyrate (bOHB) is an endogenous and specific
inhibitor of class I histone deacetylases (HDACs). Administration of exogenous bOHB, or fasting
or calorie restriction, two conditions associated with increased bOHB abundance, all increased
global histone acetylation in mouse tissues. Inhibition of HDAC by bOHB was correlated with
global changes in transcription, including that of the genes encoding oxidative stress resistance
factors FOXO3A and MT2. Treatment of cells with bOHB increased histone acetylation at the
Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1
and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with bOHB
conferred substantial protection against oxidative stress.
ellular metabolites such as acetylcoenzyme
A (acetyl-CoA) and nicotinamide adenine
dinucleotide (NAD+) influence gene expression by serving as cofactors for epigenetic
modifiers that mediate posttranslational modification of histones (1). The activity of histone
acetyltransferases (HATs) is dependent on nuclear
acetyl-CoA concentrations (2, 3) and the deacet-

1
Gladstone Institute of Virology and Immunology, San Francisco,
CA 94158, USA. 2Department of Medicine, University of California, San Francisco, CA 94143, USA. 3Gladstone Institute of
Neurological Disease, San Francisco, CA 94158, USA. 4Gladstone
Institute of Cardiovascular Disease, San Francisco, CA 94158,
USA. 5Department of Biochemistry and Biophysics, University
of California, San Francisco, CA 94143, USA. 6Sarah W. Stedman
Nutrition and Metabolism Center, and Departments of Pharmacology and Cancer Biology and Medicine, Duke University
Medical Center, Durham, NC 27704, USA. 7Laboratory of Experimental Gerontology, National Institute on Aging, National
Institutes of Health, Baltimore, MD 21224, USA. 8Department
of Chemistry, Ithaca College, Ithaca, NY 14850, USA.

*To whom correspondence should be addressed. E-mail:

everdin@gladstone.ucsf.edu

ylase activity of class III HDACs, also called sirtuins,

is dependent on NAD+ concentrations (4). Class I
(HDAC1, 2, 3, 8), class II (HDAC4, 5, 6, 7, 9,
10), and class IV (HDAC11) HDACs are zincdependent enzymes, but endogenous regulators
are not known.
Small-molecule inhibitors of class I and class
II HDACs include butyrate, a product of bacterial anaerobic fermentation (5). Butyrate is
closely related to b-hydroxybutyrate (bOHB)
(Fig. 1A), the major source of energy for mammals during prolonged exercise or starvation
(6). Accumulation of bOHB in blood increases
to 1 to 2 mM during fasting when the liver
switches to fatty acid oxidation (7, 8), and to even
higher concentrations during prolonged fasting (6 to 8 mM) (6) or in diabetic ketoacidosis
(>25 mM) (9).
To determine whether bOHB might have
HDAC inhibitor activity, we treated human embryonic kidney 293 (HEK293) cells with different amounts of bOHB for 8 hours, and measured

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Biology. Permits were approved by the Bahamian government

and the University of California, Davis. G. Mount and S. Romero
assisted with research; T. Schoener, M. Turelli, A. Hendry,
D. Nychka, L. Schmitz, and C. Boettiger provided feedback.
C.H.M. designed the study, raised funding, performed research,
collected and analyzed data, and wrote the paper. P.C.W.
contributed materials, discussed design and results, and
commented on the manuscript.

Supplementary Materials
www.sciencemag.org/cgi/content/full/339/6116/208/DC1
Materials and Methods
Figs. S1 to S9
Tables S1 to S9
References (2151)
20 July 2012; accepted 16 November 2012
10.1126/science.1227710

histone acetylation levels by Western blot with

antibodies to acetylated histone H3 lysine 9
(AcH3K9) and to acetylated histone H3 lysine 14
(AcH3K14) (Fig. 1, B and C, and figs. S1 and S2).
bOHB increased histone acetylation in a dosedependent manner, even at 1 to 2 mM, which can
occur in humans after a 2- to 3-day fast or strenuous
exercise (6, 8, 10). Like butyrate, bOHB did not
increase acetylation of a-tubulin, indicating that
it inhibits class I HDACs but not the class IIb
tubulin deacetylase, HDAC6.
To test the HDAC inhibitor activity of bOHB
and its possible selectivity, we purified recombinant human HDACs after transient transfection
of expression vectors for human epitope-tagged
(FLAG) HDAC1, HDAC3, HDAC4, and HDAC6
in HEK293T cells. We purified the HDACs,
incubated them with 3H-labeled acetylated histone H4 peptides, and measured their deacetylase
activity (Fig. 1D) (11). bOHB inhibited HDAC1,
HDAC3, and HDAC4 in a dose-dependent manner with a median inhibitory concentration (IC50)
of 5.3, 2.4, and 4.5 mM, respectively. The HDAC6
IC50 was much higher (48.5 mM) (Fig. 1E), and
bOHB did not inhibit HDAC6 activity on its
natural substrate tubulin (fig. S3). To examine
the possibility that histone acetylation was enhanced by increased concentration of acetyl-CoA
(because bOHB is catabolized into acetyl-CoA
in target tissues), we directly measured abundance of acetyl-CoA in bOHB-treated HEK293
cells, but no change was observed (fig. S4). We
also tested a possible activating effect of bOHB
on histone acetyltransferase activity of p300
and PCAF (P300/CBP-associated factor) but
did not detect any change induced by bOHB (fig.
S5). Thus, millimolar concentrations of bOHB
appear to increase histone acetylation directly
through HDAC inhibition. High concentrations
of acetoacetate (AcAc) also inhibit class I and
class IIa HDACs in vitro (fig. S6A) and in
HEK293 cells (fig. S6B). However, the concentration of AcAc in blood is one-third or less than
that of bOHB during fasting and therefore less
likely to reach concentrations that would inhibit HDACs under physiological conditions
(12). To test the relative contribution of bOHB,

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fitness maximum, suggesting that the broader topography of adaptive landscapes is more strongly
determined by stable performance constraints than
frequency-dependent dynamics.

211

acetoacetate, and acetyl-CoA to histone acetylation in response to bOHB treatment, we depleted cells of bOHB dehydrogenases (BDH1,

C
Protein Acetylation Level
(Fold)

-Hydroxybutyrate
OH
O
OH
Butyrate O

16 32

Acid Extraction

-AcH3K9

-AcH3K14
-AcLys
Coomassie

Whole Cell

100

AcH3K9
10

AcH3K14
AcTubK40

HDAC Activity
(Percent of Untreated)

OHB (mM)
0

TSA (1 M)
Butyrate
(4 mM)

OH

2) with small interfering RNA. Both enzymes

catalyze the transformation of bOHB into acetetoacetate, and their suppression had no effect on

-Ac-Tubulin

OHB (mM)

16

32

IC50
HDAC6: 48.5mM
HDAC4: 4.5mM
HDAC3: 2.4mM
HDAC1: 5.3mM

120
100
80
60
40
20
0

-Tubulin

10

OHB (mM)

30

100

Fig. 1. Inhibition of HDACs by bOHB in vitro and in vivo. (A) Structures of b-hydroxybutyrate and
butyrate. (B) Effect of bOHB, TSA, or butyrate on acetylation of histone H3 and tubulin. HEK293 cells were
treated with the indicated concentrations of drugs for 8 hours. Histones were acid-extracted, and their
acetylation was assessed by protein immunoblotting with anti-AcH3K9, anti-AcH3K14, or anti-acetyllysine
(AcLys). Proteins from whole-cell extracts were analyzed by immunoblotting with antibodies to a-tubulin
or Ac-a-Tubulin. (C) Quantification of acetylation levels from blots in (B), shown relative to untreated cells
(bOHB 0 mM). (D) Inhibition of immunopurified HDACs by bOHB in vitro. Flag-tagged HDACs were
expressed in HEK293 cells, immunoprecipitated, and incubated in vitro with a 3H-labeled acetylated
histone H4 peptide and the indicated concentrations of bOHB. HDAC activity is relative to the activity of
each enzyme without bOHB. The IC50 values of bOHB are shown.

1.6

Histone Acetylation
(Relative to Untreated)

A
OHB (mM)

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0

Histone Acetylation
(Relative to Fed)

Fed

Fasted PBS

OHB

CR

5
4
3
2
1

Fed Fast Fed Fast PBS OHB PBS OHB AL CR

K14: R2 = 0.863

5
4
3
2
1
0

0.2

0.4

0.6

0.8

1.0

1.2

AL CR

Fig. 2. Serum bOHB and global histone acetylation in kidney of mice deprived of food. (A) Serum concentration of bOHB was determined in agematched groups of three C57Bl6 mice fed or fasted (16 weeks old, fasted for
24 hours), implanted with a pump delivering either PBS or bOHB (16 weeks
old, 24 hours of pump treatment), fed ad libitum (AL) or on calorie restriction to 60% of AL
(CR) (8 months old, 6 months on CR). Mean T SE, *P < 0.05 by t test between paired conditions.
(B) Histones were purified from the kidneys of the same mice as in (A). Acetylation was assessed
with anti-AcH3K9, anti-AcH3K14, or anti-Ac-a-tubulin (fig. S8). Acetylation is normalized to total
histone H3 and shown relative to the control condition in each pair (e.g., fed versus fasted).
Mean T SE, *P < 0.05 by t test between paired conditions. (C) Serum bOHB concentrations
[from (A)] plotted against acetylation of histone H3K9 and H3K14 [from (B)]; correlation
coefficient R2 = 0.772 for H3K9 and 0.863 for H3K14.

K9: R2 = 0.772

0
1.4

1.6

OHB (mM)

212

AcH3K9 AcH3K14 AcH3K9 AcH3K14 AcH3K9 AcH3K14

AcH3K9
AcH3K14

AL

histone acetylation in response to bOHB up to

3mM. At higher concentrations of bOHB (10
and 30 mM), however, further increase in histone acetylation was suppressed by depletion
of bOHB dehydrogenases (fig. S7, A and B),
indicating that either AcAc or acetyl-CoA might
also contribute to histone acetylation in cells
exposed to concentrations of bOHB above the
IC50 for HDACs.
To determine whether changes in bOHB
concentrations in vivo might affect histone acetylation, we measured bOHB concentration in
mouse serum after a 24-hour fast or in mice
on calorie restriction (CR). We also used implanted osmotic pumps to administer exogenous bOHB or phosphate-buffered saline (PBS).
bOHB concentrations increased to 1.5 T 0.1 mM
after a 24-hour fast, 0.6 T 0.1 mM in mice on
CR and 1.2 T 0.1 mM with administration of
bOHB via an intraperitoneal pump (Fig. 2A).
We collected tissues from fed or 24-hourfasted
mice and measured histone acetylation by immunoblotting. Acetylation of H3K9 and H3K14
reflect the competing activities of HATs and
HDACs and influence gene expression in several species, including humans (13). Acetylation of H3K9 and H3K14 increased significantly
in several organs in fasted mice, particularly
kidney (Fig. 2B and figs. S8 and S9). In kidney, histone acetylation (H3K9 and H3K14) also
increased two- to fivefold in mice under CR.
Kidney histone acetylation and serum bOHB
concentrations were strongly correlated for both
histone H3K9 (R2 = 0.772) and histone H3K14
(R2 = 0.863) (Fig. 2C). We first focused on
kidney, the organ with the largest changes in
histone acetylation, to investigate the effects

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REPORTS

REPORTS

10

60

Mt2 mRNA Expression

(Relative to Untreated)

50

40

30
20

CR

OHB
AL

10
0

CR

OHB
AL

Fed

Fast
PBS

Fed

Fast
PBS

Foxo3a mRNA Expression

(Relative to Untreated)

12

PBS
OHB

7
6
5
4
3
2
1
0

10K 2K 1K 0.1K

Foxo3a
RNA (Relative to Control)

3.5
3.0

HDAC ChIP Binding

(Relative to IgG)

2K 1K 0.1K

Mt2

Foxo3a
Mt2

2.5
2.0
1.5
1.0
0.5
0

Downloaded from www.sciencemag.org on May 31, 2013

Downstream

Fig. 3. Increased expression of oxidative stress resistance genes in cells exposed to bOHB. (A) Expression of Foxo3a under various conditions (see Fig. 2
for details) measured by QPCR. Foxo3 expression is
normalized to abundance of glyceraldehyde-3phosphate dehydrogenase (GAPDH). Mean T SE,
*P < 0.05 by t test between paired conditions. (B)
Expression of Mt2, measured as in (A). (C) Promoters
of Mt2 and Foxo3a are enriched for acetylated histone H3K9 after bOHB treatment. HEK293 cells were
treated with 10 mM bOHB or PBS for 24 hours.
Chromatin was immunoprecipitated with anti-H3 or
anti-AcH3K9, and the purified DNA was analyzed with
primer pairs specific for the Foxo3a or Mt2 promoters.
Results are the ratios of AcH3K9 to total histone H3.
Mean T SE, *P < 0.05 by t test between bOHB and
PBS conditions. (D) HDAC depletion increases
Foxo3a and Mt2 mRNAs abundance. HEK293 cells
were transfected with shRNAs specific for each class I
or class II HDAC, and mRNA abundance was measured by QPCR 72 hours after transfection. Mean T
SE, *P < 0.05 by t test versus control shRNA. (E)
HDAC1, but not HDAC6, is enriched at the promoters
of Mt2 and Foxo3a. ChIP analysis of the Foxo3a and
Mt2 promoters (two primer pairs per promoter) and
Gapdh (one primer pair) from HEK293 cells with
control immunoglobulin G (IgG), anti-HDAC1, or antiHDAC6. Relative promoter binding of each HDAC is
normalized to input Gapdh. Mean T SE, *P < 0.05 by t
test versus IgG control.

promoter binding of HDAC1 (fig. S12). bOHB

appears to induce local histone acetylation at
the promoter of oxidative stress resistance genes,
Foxo3a and Mt2, by inhibiting activity of HDACs
1 and 2.
Mitochondrial superoxide dismutase (Mn-SOD)
and catalase are two other well-defined FOXO3A
targets that contribute to its protective activity
against oxidative stress (15, 17). Protein immunoblotting of kidney tissue isolated from PBS- or
bOHB-treated mice showed increased expression
of FOXO3A, Mn-SOD, and catalase (Fig. 4A and
figs. S13 and S14) .
The effect on expression of MT2, FOXO3A,
MnSOD, and catalase indicated that bOHB
might have protective activity against oxidative
stress. Carbonyl derivatives are formed by a direct metal-catalyzed oxidative attack on the
amino acid side chains of proline, arginine, lysine, and threonine. Carbonylation is irreversible
and unrepairable and accumulates as organisms
age (18). To test the possible protective role of
bOHB against oxidative stress, we implanted
mice with a subcutaneous pump delivering either bOHB or PBS for 24 hours. They then
received an intravenous injection of paraquat,
which induces accumulation of reactive oxygen species. Kidney tissue was isolated after

(AcH3 K9 /Histone)

2 (Mt2), the gene with the greatest transcriptional

response to bOHB treatment as determined by
QPCR, also protects against oxidative stress (16).
Foxo3a and Mt2 mRNA expression is also modestly increased during CR as determined by QPCR
(Fig. 3, A and B). Chromatin immunoprecipitation (ChIP) analysis of the Foxo3a and Mt2
promoters with two distinct primer pairs for
each promoter revealed increased histone H3K9
acetylation at both promoters after treatment of
HEK293 cells with a high dose of bOHB (10 mM)
(Fig. 3C).
Next, we depleted cells of each class I and II
HDAC with selective short hairpinmediated RNAs
(shRNAs). The shRNAs selective for each HDAC
suppressed expression of their cognate HDAC by
at least 60% (fig. S11). Depletion of HDAC1 or
HDAC2 caused up-regulation of Foxo3a and
Mt2 mRNA by 1.8-fold and 1.5-fold, respectively (Fig. 3D). Depletion of both HDAC1 and 2
further increased accumulation of Foxo3a and
Mt2 mRNA (Fig. 3D). ChIP analysis revealed
that HDAC1, but not HDAC6, was recruited to
the Foxo3a and Mt2 promoters and not recruited to the Gapdh promoter (Fig. 3E). Binding of HDAC1 to the Foxo3a promoter was
unchanged by bOHB treatment; thus, HDAC
catalytic activity appears not to be necessary for

Relative Promoter Acetylation

of bOHB on gene expression and cellular

phenotype.
Histone acetylation induced by HDAC inhibitors is associated with transcriptional activation
and repression of a subset of cellular genes (14).
To identify genes whose expression changed in
response to bOHB, we extracted mRNA for microarray analysis from mouse kidneys treated with
bOHB or PBS for 24 hours. As bOHB is more
abundant in fasting conditions, gene expression
changes induced by bOHB may be a subset of
those induced by fasting. Of 35,556 genes tested,
284 increased transcription in response to fasting
(false discovery rate <0.2, table S1). Four of the
five genes with the largest changes in expression
in response to bOHB were also activated in response to fasting as determined both by microarray and quantitative real-time polymerase chain
reaction (QPCR) (P < 0.001 for such overlap via
binomial distribution, tables S2 and S3). Ingenuity
pathway analysis identified two of the five bOHBinduced genes (Mt2 and Lcn2) as regulated by
FOXO3A. Overall, we found five genes in the
FOXO3A network (Foxo3a, Mt2, Lcn2, Lemd3,
and Hbp1) that had increased transcription in
response to bOHB via QPCR (fig. S10). Foxo3a,
a transcription factor, induces cell-cycle arrest and
resistance to oxidative stress (15). Metallothionein

shRNA
Control

1+2

shRNA -HDAC

25

Normal IgG
HDAC1
HDAC6

20
15
10
5
0

Primer Pair #1

Primer Pair # 2

Foxo3a

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Primer Pair # 2

Gapdh

Mt2

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D

OHB

PBS

MnSOD

B
Paraquat:

OHB

FOXO3a

PBS Pump

OHB Pump

-DNP

214

3.0

+
2.5

2.0

1.5

1.0

0.5

OHB

References and Notes

1. A. G. Ladurner, Mol. Cell 24, 1 (2006).
2. K. E. Wellen et al., Science 324, 1076 (2009).
3. H. Takahashi, J. M. McCaffery, R. A. Irizarry, J. D. Boeke,
Mol. Cell 23, 207 (2006).
4. S. Imai, C. M. Armstrong, M. Kaeberlein, L. Guarente,
Nature 403, 795 (2000).
5. E. P. Candido, R. Reeves, J. R. Davie, Cell 14, 105
(1978).
6. G. F. Cahill Jr., Annu. Rev. Nutr. 26, 1 (2006).
7. G. F. Cahill Jr. et al., J. Clin. Invest. 45, 1751
(1966).
8. A. M. Robinson, D. H. Williamson, Physiol. Rev. 60,
143 (1980).

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8
4

0
Paraquat:

PBS

OHB

Fig. 4. Protective effect of bOHB treatment against

oxidative stress. (A) Amounts of catalase, MnSOD, or
FOXO3A measured by protein immunoblotting in kidney tissue from 16-week-old mice implanted with an
osmotic pump delivering PBS or bOHB (as in Fig. 2;
n = 3); mean T SE, *P < 0.05 by t test between PBS and
bOHB conditions. (B) Protein carbonylation in kidney
samples from mice implanted with an osmotic pump
delivering PBS or bOHB (as in Fig. 2; n = 3) and treated
with paraquat (50 mg/kg) or vehicle for 2 hours. Carbonylation was measured by immunoblotting with antiDNP. All samples were run on a single gel; after imaging,
lanes were rearranged for presentation. (C) Quantification of protein carbonylation in (B). Mean T SE, *P <
0.05 by t test between PBS and bOHB conditions. (D)
Sections of kidney obtained from the same mice as in
(B) were stained with anti-4-HNE and quantified (see
fig. S16 for primary picture). Mean T SE, *P < 0.05 by t
test between PBS and bOHB conditions. (E). Lipid peroxides were quantified in mice kidneys (LPO assay kit,
Cayman, Ann Arbor, MI). Mean T SE, *P < 0.05 by t test
between PBS and bOHB conditions.

fasting and CR reveals an example of integration between metabolic status and epigenetic
changes. We show that changes in histone acetylation and gene expression caused by bOHB
promote stress resistance in the kidney. Future
studies should investigate the specific gene expression and physiological effects of bOHB in
other tissues. For example, low-carbohydrate
diets that induce substantial ketogenesis are
broadly neuroprotective and enhance resistance
of neurons to oxidative damage (20). In addition,
reduction in HDAC activity by either genetic
manipulation or chemical inhibition extends life
span in Drosophila (21, 22). Inhibition of HDACs
by bOHB might contribute to the beneficial effect of ketogenic diets and may be one mechanism by which calorie restriction confers health
benefits.

11 JANUARY 2013

16

OHB

PBS

PBS

2 hours, and protein carbonylation was assayed

by protein immunoblotting with an antibody to
dinitrophenyl (DNP) after derivatization of protein with dinitrophenylhydrazine (DNPH) (Fig.
4B). Paraquat treatment of control mice receiving
a PBS infusion led to a twofold increase in carbonylated proteins. This increase in protein carbonylation was significantly suppressed (54 T 9%
decrease) in mice receiving bOHB (Fig. 4C).
We also examined another marker of oxidative stress: lipid peroxidation. 4-Hydroxynonenal
(4-HNE) is a degradation product of polyunsaturated lipid and accumulates in response to oxidative stress (19). Kidney tissue sections from
PBS- or paraquat-treated mice were stained with
an antibody to 4-HNE, and the amount of 4-HNE
staining was quantified with imaging software
(fig. S15). Paraquat treatment increased 4-HNE
staining threefold in control mice (PBS) (Fig.
4D). This increase was completely suppressed in
mice treated with bOHB. Lipid peroxides were
also directly quantified in an enzyme-linked immunosorbent assay that measures conversion of
ferrous ions to ferric ions. A twofold increase in
lipid peroxide in response to paraquat was suppressed significantly by bOHB treatment (Fig. 4E).
Thus, bOHB protects against paraquat-induced
oxidative stress in mouse kidney.
Our observation that bOHB is an endogenous HDAC inhibitor present in organisms at
millimolar concentrations during prolonged

0
Paraquat:

OHB

0
Paraquat:

-COXV

12

Catalase

PBS

20

16

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99, 838 (2002).
Acknowledgments: We thank J. Carroll for graphics,
G. Howard and S. Ordway for editorial assistance, V. Fonseca
for administrative assistance, and A. Brunet (Stanford
University) for reagents. Supported by funds from the
Gladstone Institutes.

Supplementary Materials
www.sciencemag.org/cgi/content/full/science.1227166/DC1
Materials and Methods
Figs. S1 to S15
Tables S1 to S3
References (2326)
9 July 2012; accepted 16 November 2012
Published online 6 December 2012;
10.1126/science.1227166

www.sciencemag.org

Downloaded from www.sciencemag.org on May 31, 2013

PBS

E
20

Lipid Peroxide
(nmol/mg)

HNE Staining
(Percent of Area)

3.0
2.5
2.0
1.5
1.0
0.5
0

Relative Protein Carbonylation

Protein Expression
(Relative to Untreated)