SIRTUIN 1 AND SIRTUIN 3: PHYSIOLOGICAL
MODULATORS OF METABOLISM
Ruben Nogueiras, Kirk M. Habegger, Nilika Chaudhary, Brian Finan, Alexander S. Banks,
Marcelo O. Dietrich, Tamas L. Horvath, David A. Sinclair, Paul T. Pfluger, and Matthias H. Tschöp
Department of Physiology, School of Medicine-Instituto de Investigaciones Sanitarias, University of Santiago de
Compostela, Santiago de Compostela, Spain; CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn), Santiago de
Compostela, Spain; Metabolic Diseases Institute, Department of Medicine, University of Cincinnati, Cincinnati, Ohio;
Institute for Diabetes and Obesity, Hemholtz Center Munich and Technical University Munich, Munich, Germany;
Department of Cancer Biology and Division of Metabolism and Chronic Disease, Dana-Farber Cancer Institute,
Boston, Massachusetts; Program on Cell and Neurobiology of Energy Metabolism, Section of Comparative Medicine,
Yale University School of Medicine, New Haven, Connecticut; Department of Obstetrics, Gynecology and
Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut; Department of Neurobiology,
Yale University School of Medicine, New Haven, Connecticut; Department of Pathology and Paul F. Glenn
Laboratories for the Biological Mechanisms of Aging, Harvard Medical School, Boston, Massachusetts
sirtuin 3 (SIRT3), which are localized to the nucleus and mitochondria, respectively. Both are
activated by high NAD?levels, a condition caused by low cellular energy status. By deacetylating a
variety of proteins that induce catabolic processes while inhibiting anabolic processes, SIRT1 and
SIRT3 coordinately increase cellular energy stores and ultimately maintain cellular energy homeo-
stasis. Defects in the pathways controlled by SIRT1 and SIRT3 are known to result in various
metabolic disorders. Consequently, activation of sirtuins by genetic or pharmacological means can
elicit multiple metabolic benefits that protect mice from diet-induced obesity, type 2 diabetes, and
nonalcoholic fatty liver disease.
Nogueiras R, Habegger KM, Chaudhary N, Finan B, Banks AS, Dietrich MO, Horvath
Metabolism. Physiol Rev 92: 1479–1514, 2012; doi:10.1152/physrev.00022.2011.—
The sirtuins are a family of highly conserved NAD?-dependent deacetylases that act as
cellular sensors to detect energy availability and modulate metabolic processes. Two
sirtuins that are central to the control of metabolic processes are mammalian sirtuin 1 (SIRT1) and
THE STRUCTURE, FUNCTION, ...
CELLULAR FUEL SENSING BY SIRT1 ...
MOLECULAR TARGETS OF SIRT1 ...
ENDOGENOUS REGULATORS OF...
METABOLIC TISSUES TARGETED ...
METABOLIC TISSUES TARGETED ...
GENETIC MODELS OF SIRT1 ...
GENETIC POLYMORPHISMS OF SIRT1 ... 1502
METABOLIC CONSEQUENCES OF ...
cellular energy metabolism are nearly identical. In all living
organisms, cellular energy is produced and expended using
highly homologous pathways, and energy is stored and trans-
ferred using universal “energy currencies” such as ATP and
NADH. The tight balance between such anabolic and cata-
bolic pathways ensures that cells do not deplete essential en-
ergy stores, which would ultimately cause cellular damage or
death. Accordingly, evolutionarily conserved mechanisms
have been established to protect cells from low cellular energy
availability and to store excess energy for future use. Such
mechanisms include the mTOR signaling pathway to detect
branched-chain amino acids like leucine, carbohydrate re-
sponsive element binding protein (ChREBP) to respond to
sense low cellular ATP levels, ghrelin O-acetyltransferase
(GOAT) to detect medium-chain fatty acids, and sirtuins to
detect NAD?/NADH levels. This review focuses on SIRT1
malian energy homeostasis, and gives an overview on the
manyfold metabolic benefits elicited by both fuel sensors.
A. Historical Background
Almost a century ago, the Nobel laureate Francis Peyton
Rous reported that chronic calorie restriction elicits benefi-
cial metabolic effects on the spontaneous occurrence of tu-
mors in rats (279). At the same time, Osborne et al. (238)
showed that calorie restriction of young rats (1.5–6 mo of
age) restores fertility at a later age and prolongs life span
Physiol Rev 92: 1479–1514, 2012
(238). While initially these findings were widely ignored, a
growing number of studies in the following decades corrob-
orated these benefits of calorie restriction, and accumulated
evidence demonstrated that calorie restriction also protects
from other age-related diseases, such as chronic kidney fail-
ure. In 1960, Berg and Simms (38) proposed that the reduc-
tion in body fat plays a decisive role in mediating the ben-
eficial effects of calorie restriction on fertility, age-related
due to a decrease in the absolute amount of calories con-
sumed (40). More recently, this concept has been chal-
lenged (211), and evidence points to the effect of specific
amino acids in determining the effects of long-term calorie
restriction on life span and fertility (118, 219). In the past
two decades, new techniques made it possible to focus also
on the molecular underpinnings of metabolic benefits
through calorie restriction. Several mechanisms have been
identified that were shown to play a role, such as the atten-
uation of oxidative damage, decreases in insulin and glu-
cose levels, an impairment of the growth hormone-insulin-
like growth factor I (IGF-I) axis, and an activation of the
deacetylase family of sirtuins.
In parallel with the work on calorie restriction, a genetic
screen for long-lived mutants of Saccharomyces cerevisiae
led to the discovery that the silent information regulation-2
(Sir2) gene could slow aging in this species (164, 306). Sir2
was later identified as NAD?-dependent deacetylase for hi-
stone proteins that is required for calorie restriction to ex-
tend yeast life span (150, 203). Further studies identified
structural and functional homologs in numerous other or-
ganisms (217). This review summarizes current research on
metabolic effects elicited by pharmacological, genetic, or
physiological manipulation of sirtuins.
The mammalian sirtuins are a family of NAD?-dependent
enzymes with homology to the Saccharomyces cerevisiae
gene silent information regulator 2 (Sir2). Humans have
seven sirtuins, SIRT1-SIRT7. SIRT1, the most studied
member of this family, plays an important role in several
processes ranging from cell cycle regulation to energy ho-
meostasis. SIRT3 has recently emerged as a sirtuin with
considerable impact on mitochondrial energy metabolism
and function. Numerous studies have also demonstrated
that both SIRT1 and SIRT3 play an important role in dif-
ferent types of cancer. SIRT1 has tumor suppression activ-
ity in ageing- and metabolic syndrome-associated cancer
(123, 133, 134, 283), and SIRT3 has also been identified as
a tumor suppressor (35, 100, 171, 245).
The basic sirtuin structure and function have remained
highly conserved across species, from bacteria to humans.
In humans, sirtuins exist throughout the body; for example,
SIRT1 is expressed in the brain, liver, pancreas, adipose
tissue, muscle, and heart. Sirtuins may function as deacety-
residues of numerous target proteins, including histones
and transcription factors. Section II details the structure,
function, and localization of sirtuins.
Sirtuins are cellular energy sensors that require NAD?for
their enzymatic activity. As a result, their activity is directly
linked with metabolism. Certain cellular stressors or a low
energy state in the cell increases the NAD?/NADH ratio,
decreases nicotinamide levels, and activates sirtuins (13,
for sirtuin-catalyzed NAD?-dependent reactions.
Mammalian SIRT1 deacetylates a host of target proteins
that are important for apoptosis, the cell cycle, circadian
rhythms, mitochondrial function, and metabolism. In par-
ticular, much current research focuses on the impact of
SIRT1 in glucose homeostasis, lipid metabolism, and en-
ergy balance. While SIRT1 plays an important role in met-
abolic function, sirtuins 3–5 are localized in mitochondria
and may regulate mitochondrial energy metabolism.
SIRT3, the most studied of the mitochondrial sirtuins,
deacetylates a number of mitochondrial proteins and might
also play a role in regulating ATP production. Section IV
focuses on the molecular targets of SIRT1 and SIRT3.
Mammalian sirtuins are not only regulated by NAD?/
NADH ratio or cellular stressors, but also by endogenous
proteins involved in signal transduction and transcription,
as well as by a number of microRNAs. Section V will de-
scribe the complex regulation of SIRT1 and SIRT3 by en-
Sirtuins 1 and 3 are expressed in a wide variety of tissues
and target numerous proteins. Section VI depicts how the
activation of SIRT1 influences metabolically active tissues,
such as liver, skeletal muscle, pancreas, adipose tissue, or
brain, by inducing a wide range of physiological processes.
Section VII focuses on the specific roles of SIRT3 in diverse,
metabolically active tissues.
Several mouse models have been used to characterize the
metabolic functions of sirtuins. Inbred whole body SIRT1
knockout mice are born underweight and do not live past
the early postnatal stage. SIRT1 knockout mice on an out-
bred background exhibit developmental and metabolic ab-
normalities including cardiac defects and decreased loco-
motor activity, but they also exhibit improved glucose ho-
meostasis. Several studies focus on liver-specific ablation of
SIRT1 and the role of hepatic SIRT1; however, many of
these findings are contradictory. In addition to SIRT1 defi-
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ciency, SIRT1 overexpression is also under investigation.
Mice with global SIRT1 overexpression show resistance to
metabolic dysfunction as a result of high-fat diet exposure.
Gain- and loss-of-function studies on sirtuins other than
SIRT1 have also been performed. Of particular importance
to metabolic regulation are the mitochondrial sirtuins. Sec-
tion VIII outlines the metabolic phenotypes of global or
tissue-specific SIRT1 and SIRT3 loss- and gain-of-function
models and depicts specific roles of SIRT1 for circadian
rhythms and diabetes-induced cardiac function.
Section IX describes the current knowledge on genetic poly-
morphisms in SIRT1 and SIRT3 and their implication in
It has been suggested that pharmacological SIRT1 activation
other metabolic disorders. Resveratrol is a proposed SIRT1
activator, but its metabolic benefits, when administered phar-
macologically in humans, are still a matter of controversy.
Several small-molecule SIRT1 activators with improved phar-
consequences of SIRT1 activation by resveratrol and other
natural or synthetic ligands.
Finally, section XI gives a general view on future directions
and perspectives for sirtuin research.
II. THE STRUCTURE, FUNCTION,
AND DIVERSITY OF SIRTUINS
A. Sirtuins Are Highly Conserved
The Saccharomyces cerevisiae gene silent information reg-
ulator 2 (Sir2) was identified as a NAD?-dependent histone
deacetylase (150, 309) involved in life span extension asso-
ciated with calorie restriction (13, 201). Sir2 homologs,
known as sirtuins, have been identified in numerous higher
organisms including Drosophila melanogaster (31, 231),
Caenorhabditis elegans (327), mice (366), and humans (51,
107, 108, 298). Seven sirtuins (SIRT1-SIRT7) comprise the
sirtuins share the conserved sirtuin domain, but vary in
subcellular localization and function. Of note, sirtuins have
been lost in many species including insects, nematodes, and
plants (121). Thereby, it seems that the loss of individual
sirtuins might be compensated for by redundant functions
in remaining sirtuin family members (121).
In yeast, Sir2 deacetylates the acetyl-lysine residues of his-
tones (150) by catalyzing a unique chemical reaction that
requires NAD?and generates the novel product O-acetyl-
ADP-ribose (O-AADPR) (48, 72, 321, 324). As discussed
below, this deacetylase activity is conserved in mammalian
sirtuins. In addition to histone deacetylation, mammalian
sirtuins are also able to catalyze reactions for a number of
protein substrates (107). Furthermore, certain mammalian
sirtuins possess ADP-ribosyltransferase activity (107).
and its homologs, increased protein levels of Sir2 not only
to C. elegans (327) and D. melanogaster (277). However,
other lines of evidence suggest that Sir2 does not affect life
span in flies (231). Elevated expression of sirtuins in normal
human cells does not extend replicative life span (218), and
there is no evidence that a sirtuin can extend the life span of
a mammal, unless it is under metabolic stress (34, 246).
Today, we know that biochemical features are highly con-
served in sirtuins. Their physiological roles, however, differ
deacetylation of histones, but also controls segregation of
protein carbonylation, mammalian sirtuins target multiple
proteins, regulating many diverse processes ranging from
cell cycle progression and mitochondrial function to metab-
olism and energy homeostasis.
B. Structural Properties of SIRT1
Among the yeast proteins that comprise the Sir silencing
complex (Sir1–4), Sir2 is unique (reviewed in Ref. 117), as
it is the only homolog that has been evolutionarily con-
served from bacteria to humans (3, 51, 107, 298). Accord-
ingly, all Sir2-like proteins possess a sirtuin core domain
containing a series of sequence motifs that is conserved
across organisms (51). Molecular phylogenetic analyses of
a diverse array of organisms (including archaea bacteria,
the conserved sirtuin core domain sequences of eukaryotic
organisms can be grouped into four main classes: SIRT1,
SIRT2, and SIRT3 compose class I, SIRT4 composes class
II, SIRT5 composes class III, and SIRT6 and SIRT7 com-
pose class IV (108). Sir2 protein is also unique among the
silencing factors in Saccharomyces cerevisiae because it si-
lences the rDNA as well as the silent mating-type loci and
telomeres (303). Silencing is a universal form of transcrip-
tional regulation in which regions of the genome are revers-
ibly inactivated by a myriad of mechanisms. Sir2 is also
thus sparing the daughter cell (4). However, the molecular
target of this action and how this relates to mammalian
aging is not yet known.
ing, DNA repair, and life span extension by calorie restriction
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are ?30–65% identical to Sir2 and are characterized by a
conserved core domain, which is up to 84% identical to Sir2
and essential for Sir2 silencing (298).
To understand the structural basis of the enzymatic mech-
anism of the sirtuins, a number of crystal structures have
been determined. In Archaeoglobus fulgidus, two crystal
structures of Sir2-Af1 complexed with NAD were solved at
2.1 and 2.4 Å resolution (101, 221). The structure revealed
that the protein consists of a large domain having a Ross-
mann fold and a small domain containing a three-stranded
zinc ribbon motif. NAD is bound in a pocket between the
twodomains(221) (FIGURE 1).Ontheotherhand,the1.7Å
crystal structure of the 323-amino acid catalytic core of
human SIRT2 reveals an NAD-binding domain, which is a
variant of the Rossmann fold, and a smaller domain com-
posed of a helical module and a zinc-binding module. Mu-
tagenesis studies suggest that a conserved large groove at
the interface of the two domains is the likely site of catalysis
(101). More recently, several crystal structures for human
SIRT3 have been also reported (160), including the apo-
structure without substrate, a structure with a peptide con-
taining acetyl lysine of its natural substrate trapped by a
thioacetyl peptide, and a structure with the dethioacety-
lated peptide bound (160) (FIGURES 2 and 3). Finally, the
dle that in other sirtuin structures connects the zinc-binding
motif and Rossmann fold domain (243). SIRT6 also lacks the
conserved, highly flexible, NAD?-binding loop and instead
contains a stable single helix (243).
C. Tissue Distribution of SIRT1
has been detected in the liver, pancreas, heart, muscle, and
adipose tissue of mice (151, 191, 374). Other reports, uti-
lizing a mCherry reporter gene in C. elegans, identified Sir2
mainly in muscle and intestinal cells (27). In humans and
Zn2+ Binding Site
in the cartoon representation with the zinc binding site highlighted in cyan, the helical module highlighted in
magenta, and the Rossmann foldlike NAD?binding site highlighted in orange. The acetyl-ADP intermediate is
depicted as a stick representation with carbon atoms in gray, oxygen atoms in red, nitrogen atoms in blue,
sulfur atoms in yellow, and phosphate atoms in orange. The structure was assembled from Protein Data Bank
code 2HJH (Protein Data Bank: Hall BE, Buchberger JR, Gerber SA, Ambrosio ALB, Gygi SP, Filman D, Moazed
D, Ellenberger T.).
Structure of yeast Sir2 in complex with an acetyl-ribosyl-ADP intermediate. Yeast Sir2 is depicted
Zn2+ Binding Site
toon representation with the zinc binding site highlighted in cyan, the
helical module highlighted in magenta, the Rossmann foldlike NAD?
binding site highlighted in orange, and the domain interface loops
highlighted in white. The structure was assembled from Protein
Data Bank code 3GLS (Jin et al., Ref. 160).
Structure of apo SIRT3. SIRT3 is depicted in the car-
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mice, SIRT1 is ubiquitously expressed in the brain, espe-
cially in areas related to neurodegenerative diseases, such as
the prefrontal cortex, hippocampus, and basal ganglia
(379). SIRT1 is also expressed in important metabolic cen-
ters of the brain (268), including the hypothalamic arcuate,
the area postrema and the nucleus of the solitary tract in the
hindbrain. During aging, SIRT1 expression is decreased in
specific nuclei of the hypothalamus of mice, but not in all
brain regions (185). Other studies focusing on the function
of SIRT1 in development found that SIRT1 is expressed at
high levels in most of the tissues of embryos, where expres-
sion was found in the heart and the brain, and to a lesser
extent in liver, spleen, kidney, lung, thymus, testis, and
ovary (280). Overall, these studies suggest that SIRT1 is
expressed in a wide variety of tissues.
D. Subcellular Distribution of SIRT1
The subcellular localization of SIRT1 likely depends on cell
type, stress status, and molecular interactions. SIRT1 has
it interacts with both nuclear and cytosolic proteins (62,
218, 322). For example, SIRT1 is ubiquitously present in
the prefrontal cortex, hippocampus, and basal ganglia.
Throughout the rodent brain and spinal cord, both cytoso-
lic and nuclear localization can be detected, although im-
munocytochemical and Western blot analyses indicate that
SIRT1 is predominantly nuclear (379). The primarily nu-
clear localization of SIRT1 can be attributed to two nuclear
localization signals and two nuclear export signals (322).
Thus the extent of cytosolic versus nuclear localization of
SIRT1 may be dictated by the relative strengths of the nu-
clear localization and export signals in each cell type or
environmental signal (123).
E. Tissue Distribution of SIRT3
Like SIRT1, mammalian SIRT3 is expressed in a variety of
tissues. Studies carried out with quantitative RT-PCR in dif-
ferent mouse tissues show the highest expression in kidney,
brain, and heart, followed by liver and testes, with lower ex-
pression in lung, ovary, spleen, and thymus (159). Similar
expression patterns are seen in human tissues (314, 354).
F. Subcellular Distribution of SIRT3
SIRT3 was the first mammalian sirtuin shown to be local-
ized to mitochondria (218, 236, 294). It is localized to the
mitochondrial matrix, and cleavage of its signal sequence is
necessary for enzymatic activity (294). The major isoform
of mouse SIRT3 is a 257-amino acid protein that aligns
with the COOH-terminal portion of human SIRT3 (resi-
dues 143–399) (366). However, subsequent reports have
questioned the exclusivity of the mitochondrial distribution
of SIRT3. This ambiguity likely arises from the two differ-
ent isoforms (short and full length) of SIRT3, which are
expressed in both humans and mice and appear to be dif-
ferentially distributed in a tissue-specific manner (124, 253,
319). Additionally several splice variants (M1, M2, and M3)
have been reported (64). The variants M1 and M2 appear to
be localized to the mitochondria, whereas the M3 splice vari-
SIRT3 has been reported to be exclusively localized to the
mitochondria (159), other groups report that in the heart
cytoplasm, and the nucleus (319). However, after overexpres-
sion of short and long forms of mSIRT3 in mouse embryonic
fibroblasts and H9C2 cells, both forms were located in the
nucleus as well as the cytoplasm (29).
As with the mouse SIRT3 protein, there are also two forms
of the human SIRT3 protein. hSIRT3 is found in both long
full-length form is localized exclusively to the mitochon-
dria, whereas the short form, which loses the NH2-terminal
142 residues, is present in the cytoplasm and nucleus (65).
However, recent findings have questioned whether the lo-
calization of human SIRT3 is exclusive to mitochondria
(65, 236, 289). One study now suggests that SIRT3 is pres-
ent in the nucleus under basal conditions, but translocates
to the mitochondria during cellular stress (289). Indeed, the
Zn2+ Binding Site
ADPR. SIRT3 is depicted in the cartoon representation with the zinc
binding site highlighted in cyan, the helical module highlighted in
magenta, and the Rossmann foldlike NAD?binding site highlighted in
orange. The acetyl-lysine substrate, which here is the trapped reac-
tion intermediate AceCS2-Ks-acetyl-ADPR peptidal compound, in-
serts into the domain interface populated by loops that are high-
lighted in white where catalysis of the deacetylation occurs. The
AceCS2-Ks-acetyl-ADPR is depicted as a stick representation with
carbon atoms in gray, oxygen atoms in red, nitrogen atoms in blue,
sulfur atoms in yellow, and phosphate atoms in orange. The struc-
ture was assembled from PDB code 3GLT (Jin et al., Ref. 160).
Structure of SIRT3 in complex with AceCS2-Ks-acetyl-
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location of SIRT3 in the nucleus is a matter of controversy,
subcellular fractionation failed to find hSIRT3 in the nu-
cleus, suggesting it is exclusively mitochondrial (65). The
distinct methodologies and antibodies used by the different
laboratories might explain these differences (65).
III. CELLULAR FUEL SENSING BY SIRT1
Sirtuins function as cellular energy sensors that are either ac-
tivated or inhibited by the level of metabolic cofactors and
intermediates in the cell, including NAD?, NADH, and nico-
produces energy for use in cellular metabolism is the mito-
chondrion. Mitochondria are equipped with machinery to fa-
cilitate the oxidation of substrate (e.g., carbohydrates and
energy, which is stored in reduced carrier molecules (as
FADH2and NADH) that are utilized in the mitochondrial
membrane to generate ATP through the oxidative phosphor-
ylation cascade. Thus the generation of energy (i.e., ATP) in
the cells is linked to the production of FADH2and NADH.
When these carriers are oxidized, they become FAD and
NAD?, and all four molecules are important regulators of
numerous biochemical reactions inside the cell.
A. Cellular Energy: NAD?/NADH Ratios
and the NAD?Salvage Pathway
strate NAD?, which places the sirtuins at the nexus of cel-
lular energy metabolic regulation and provides a possible
link between cytosolic energy status and nuclear signaling.
Catabolic reactions such as ?-oxidation, glycolysis, protein
degradation, and citric acid cycling reduce NAD?to
NADH. When energy is plentiful, intracellular NADH lev-
els rise while NAD?levels drop, although the ratio always
favors NAD?over NADH (232). NADH from cytosolic
metabolism feeds into the mitochondria through the
malate-aspartate shuttle or by reducing pyruvate to lactate
to join NADH produced via the citric acid cycle (25). Re-
gardless of its origin, NADH is oxidized in the mitochon-
dria by the electron transport chain (ETC). Specifically,
NADH feeds into the ETC through its oxidation to NAD?
at complex I (137). Subsequent reactions of the ETC pro-
duce energy that is used to pump protons across the mito-
chondrial matrix. This generates the proton gradient that
drives ATP synthesis via oxidative phosphorylation.
Cellular NAD?is derived from two main sources: de novo
synthesis from the amino acid tryptophan (215) and via the
salvage of nicotinamide back to NAD?. The product of the
sirtuin reaction, nicotinamide, acts as a feedback inhibitor
by binding in a conserved regulatory pocket on the enzymes
called the C-pocket (20, 42).
In yeast, the removal of nicotinamide, the rate-limiting step
of the NAD salvage pathway, is catalyzed by pyrazinami-
dase/nicotinamidase 1 (PNC1), a nicotinamide deaminase
that is upregulated in response to caloric restriction and
cellular stress (13). Overexpression of NAD salvage genes
extends life span in a Sir2-dependent manner (12), while
deletion of the PNC1 gene blocks life span extension by
calorie restriction (13). These findings are consistent with
the hypothesis that the ability of dietary manipulations to
extend life span in mammals is mediated, in part, by the
induction of enzymes that increase the rate at which nico-
tinamide is recycled back to NAD?(361).
In humans, the rate-limiting step of the NAD?salvage path-
way is catalyzed, not by PNC1, but by a nicotinamide phos-
known as PBEF), which converts nicotinamide to nicotin-
in times of prolonged fasting (362). Additional findings sug-
gest that NAMPT could directly influence the NAD?/NADH
al. (273) suggest that overexpression of NAMPT directly in-
creases SIRT1 activity, implicating NAD?concentration in
the regulation of SIRT activity.
Studies in yeast bolstered the hypothesis that the NAD?/
NADH and the NAD?/nicotinamide ratios regulate sirtuin
activity. Calorie restriction induces a decrease in both
NADH and nicotinamide concentrations and is associated
with increased life span and Sir2 activity (13, 201–203,
286). Despite strong genetic evidence, a subsequent bio-
chemical study questioned whether NADH is in sufficient
concentrations in vivo to inhibit sirtuins. This study sug-
gested that the ratio of NAD?to nicotinamide is the pri-
mary mechanism of sirtuin regulation (291).
Studies in mice suggest that prolonged fasting can induce a
50% increase in hepatic NAD?, which was associated with
increased SIRT1 activity (275). Similarly, Yang et al. (362)
demonstrated that food deprivation increased the levels of
NAMPT and mitochondrial NAD?levels, and that the ensu-
ing stress protection required both SIRT3 and SIRT4. Indeed,
the dependency of mitochondrial SIRT3 on NAD?has been
clearly demonstrated in multiple studies (236, 289, 294).
B. Mechanism of NAD?-Dependent
The Sir2 family of class III deacetylases utilizes a distinctive
chemical reaction to deacetylate lysine residues. This reac-
tion consumes NAD?to produce nicotinamide, the
deacetylated substrate, and O-AADPR (48, 72, 321, 324).
The NAD?-dependent deacetylation reaction is a six-step
reaction eloquently reviewed by Sauve (284). Briefly, the
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reaction begins with the binding of NAD?to the sirtuin
catalytic site, a process mediated by highly conserved cata-
lytic phenylalanine (129, 138) and histidine residues (221,
285, 307). The first step in the sirtuin-catalyzed deacetyla-
tion utilizes amide cleavage of NAD?to produce nicotin-
amide and a peptidylimidate intermediate (285). This is
followed by formation of a 1=,2=-acyloxonium via nucleo-
philic attack of the 2=-OH group of the imidate intermedi-
ate. This collapse is facilitated by the conserved catalytic
histidine (221, 307), which acts as an activating base to the
lysis to 2=-AADPR via attack by water, releasing the free
amino group of the previously acetylated lysine residue,
AADPR, and nicotinamide. A caveat of this efficient reac-
tion is its inhibition by the reaction product nicotinamide
(42). This product can bind to the active site, reacting with
peptidyl imidate to form the reverse reaction yielding
NAD?. In addition to the sirtuin-mediated deacetylation, a
spontaneous isomeric transition occurs in the solvent to
maintain equilibrium of 2=- and 3-AADPR (285).
C. Mechanism of NAD?-Dependent
In addition to the more widely studied deacetylation reac-
tion, sirtuin enzymes were first identified by their ability to
catalyze ADP-ribosylation of target proteins (107, 323).
While the mechanisms of ADP-riboslytransfer are still in
for this reaction (98, 129, 130, 284). A leading hypothesis
by Hawse and Wolberger (130) suggests that the targets of
sirtuin-mediated ADP-riboslytransfer contain a nucleo-
philic residue ?2 positions from the acetylated lysine. In
this mechanism, NAD?reacts with acetyllysine to form an
O-alkyamidate and release nicotinamide. The ?2 nucleo-
philic residue next attacks the O-alkylamidate intermediate
to produce ADP-ribosylation and acetyllysine (130). A re-
cent study on SIRT6 indicates that this sirtuin physically
associates with poly-ADP-ribose polymerase 1 (PARP1),
mono-ADP-ribosylates, and PARP1 on lysine residue 521,
thereby stimulating PARP1 poly-ADP-ribosylase activity
and enhancing double-strand break repair under oxidative
D. Regulation of Sirtuin Activity by Calorie
Restriction and Metabolic Stressors
Although some exceptions exist, studies in the area of aging
have shown repeatedly that caloric restriction (CR) im-
proves glucose metabolism, increases mitochondrial activ-
ity, and extends life span in a broad range of species from
yeast to mammals (66, 127, 132, 144). In yeast, a reduction
of glucose from 2 to 0.5% in the media extends life span
?30%. Furthermore, Sir2 and respiration are required for
this effect (13, 187, 203).
However, the benefits of CR in yeast are not solely dependent
on Sir2. Severe CR (0.05% glucose) extends yeast replicative
life span but does not require Sir2 nor mitochondrial respira-
under starvation (97) or in worms under CR (41).
It is important to point out that the amount of yeast Sir2
protein does not increase during CR (13), and there are
alternative hypotheses concerning the mechanism for CR-
mediated longevity. Surprisingly, Sir2 levels and NAD?
concentrations are not elevated following CR in yeast (12).
Instead, enzymatic activity is believed to increase in re-
sponse to changes in the concentrations of nicotinamide
and NADH, where PNC1 plays an important role as de-
SIRT1 appears to be the one that most closely resembles the
yeast Sir2 enzyme (108). CR studies in rodent models have
shown that, unlike its effect in yeast, CR increases SIRT1 in
several metabolically relevant tissues including brain, kid-
ney, liver, white adipose tissue, and skeletal muscle (63).
The CR-stimulated increase in SIRT1 protein is accompa-
nied by increased NAD?, suggesting elevated activity in
link between CR and SIRT1, analyses of genetically ele-
vated SIRT1 suggest phenotypes that are highly reminiscent
of those displayed during treatment with CR and SIRT1
agonists (34, 186, 220). Nevertheless, the hypothesis that
SIRT1 activity is increased in all tissues after CR has been
called into question recently by a report showing SIRT1
activity in the liver is decreased by CR, and that this reduc-
tion by CR is correlated with the reduced role of this organ
in fat synthesis (74). However, the time of day, species, and
type of CR are all variables that may explain differences
between these studies.
Overexpression of SIRT1 in mice is associated with positive
metabolic outcomes. Specifically, these mice display a de-
crease in adiposity, serum cholesterol, and insulin, while
displaying increased resistance to obesity-generated glucose
intolerance and insulin resistance (28, 46). On the other
hand, studies utilizing mice deficient for the SIRT1 gene
have also provided insight into its role in the physiology of
CR (5). Specifically, the global SIRT1-deficient mice in a
mixed background elicit a shortened life span, which is
resistant to CR (75, 196). Moreover, SIRT1-deficient mice
in a clean background are perinatally lethal, and therefore,
In addition to its activation during CR, SIRT1 is also acti-
activity is important in the context of tissue preservation.
For example, the SIRT1-mediated deacetylation of FOXO
appears to protect ?-cells from oxidative stress (174, 175).
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Similarly, while HFD exposure induces oxidative stress via
NF-?B (58), SIRT1 overexpression has been shown to in-
crease antioxidant defense enzymes and inhibit NF-?B ac-
tivation, thereby providing powerful protection from he-
patic inflammation, glucose intolerance, and NAFLD
(249). SIRT1 has been also proposed as a link between
caloric intake and mood. Specifically, in conditions of CR,
when SIRT1 is activated, anxious behavior is increased (93,
Likewise, the effect of SIRT1 in neuronal tissues appears to
be protective in the context of oxidative stress. Recent stud-
ies have suggested that SIRT1 is responsible for protection
from neuronal, and specifically axonal, degeneration (16,
be important in the treatment of Alzheimer’s disease or
other neurodegenerative diseases such as Huntington’s dis-
from oxidative damage, work by several groups suggests
that SIRT1 may also have a role in providing genetic stabil-
ity during times of oxidative damage. Specifically, SIRT1
deacetylates several genes important for DNA repair (377,
growth arrest (295). Additionally, SIRT1 is directly re-
cruited to sites of broken DNA and helps recruit key DNA
repair proteins such as Rad51 and Nbs1 (235). Further
supporting a role in genetic stability, SIRT1?/?embryos
exhibit impaired DNA repair and increased chromosomal
abnormalities (348), while overexpression of SIRT1 re-
duces aneuploidy and delays lymphoma (235).
E. Regulation of SIRT3 Activity by Nutrient
Intake and Metabolic Stressors
During CR, expression of SIRT3 is increased in the mito-
white and brown adipose tissues (241, 300, 319). This is of
interest as SIRT3 deacetylates, and therefore activates, the
enzyme responsible for formation of acetyl-CoA from ace-
tate [acetyl-CoA-synthase (AceCS)] (293). AceCS is critical
during times of prolonged fasting, where the acetate must
first be converted to acetyl-CoA before it can be utilized as
a metabolite in the citric acid cycle. Conversely, in geneti-
cally obese mice, SIRT3 and genes important for mitochon-
drial function show decreased expression in brown adipose
IV. MOLECULAR TARGETS OF SIRT1 AND
SIRT1 interacts with and regulates a number of histone and
non-histone protein substrates (FIGURE 4). The wide variety
of endogenous targets is correlated with the different bio-
logical functions modulated by this deacetylase. It has roles
in developmental and aging regulation, energy metabolism,
inflammation, and the repair of DNA double-strand break,
A. Cytosolic and Nuclear Targets of SIRT1
Histones are proteins that are essential for the tight pack-
aging of DNA into chromatin. The level of histone acetyla-
tion has been shown to regulate gene transcription. Specif-
ically, increased acetylation prevents histones from binding
and condensing DNA, thus enabling more transcription,
whereas deacetylation promotes histone binding and de-
creases transcription. Each year, different reports identify
new histones deacetylated by SIRT1, including histone 1 on
lysine 27 (H1K27) (184); histone 3 on lysines 9, 14, 18, and
56 (H3K9, H3K14, H3K18, H3K56) (150, 337, 376); and
histone 4 on lysines 12 (H4K12) and 6 (H4K6) (109, 150,
338). The molecular mechansims that target SIRT1 to its
substrates are not well understood. This lack of significant
substrate specificity has increased the difficulty in identify-
is given to studies wherein gain and loss of SIRT1 function
have reciprocal effects on substrate acetylation.
SIRT1 has been shown to target and deacetylate the lysine
residues of not only histones but numerous protein sub-
strates as well. This diversity of function has important
implications for mammalian cell survival and senescence.
The substrates of SIRT1 that regulate apoptosis and cell
cycle regulation include transcription factors and DNA re-
pair factors. p53 is an important tumor-suppressing protein
that regulates many cellular activities, such as cell cycle
regulation, DNA repair, and programmed cell death. As
SIRT1 binds and deacetylates p53 to decrease its transcrip-
tional activity, p53 is suggested to play a central role in
SIRT1-mediated functions in tumorigenesis and senescence
(78, 189, 208, 339). For this reason, SIRT1 was originally
considered to be a potential tumor promoter. However,
new evidence suggests that SIRT1 acts as a tumor suppres-
sor based on its role in negatively regulating ?-catenin and
survivin (370). Therefore, even though the interaction be-
tween SIRT1 and p53 is clear, the current role of the
SIRT1/p53 pathway on tumorigenesis is controversial.
Recent evidence suggests that the interaction between
SIRT1 and p53 is regulated by the methyltransferase
Set7/9, since the presence of the methyltransferase Set7/9
suppresses this interaction (204). Set7/9 is a SET domain-
containing histone 3 lysine 4 (H3-K4) methyltransferase
(234, 344). Set7 is known to stimulate activator-induced
transcription in vivo (234), and one of these transcription
factors might be p53.
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3. DNA damage proteins: Ku70, FOXL2, NBS1,
WRN, and XPC
Ku70 is one of the two subunits (Ku70 and Ku80) of the Ku
protein. The Ku protein plays a key role in multiple nuclear
processes, including DNA repair, chromosome mainte-
nance, transcription regulation, and recombination (272).
It has been reported that SIRT1 prevents apoptosis by in-
teractions with Ku70 (15, 49, 63, 157, 197). In response to
CR, SIRT1 deacetylates Ku70, which in turn reduces stress-
tor, from mitochondria (63). SIRT1 can also bind and
deacetylate Ku70 to increase DNA repair activity in cells
subjected to radiation exposure (157).
When the transcription factor FOXL2 is upregulated, it
promotes cell accumulation in G1phase and protects cells
from oxidative damage (254). SIRT1 inhibition increases
both amount and activity of FOXL2, thereby limiting pro-
repair factor, and its phosphorylation delays cell cycle pro-
gression (116). SIRT1 deacetylates NBS1 and thereby it is
Heat shock proteins
Inflammation and neuroprotection
Fatty acid oxidation
Cholesterol and lipogenesis
Oxidative stress and apoptosis
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one of the suggested pathways to regulate cell survival
Werner Syndrome (WRN) is a gene with essential functions
in maintaining genome stability. After DNA damage,
SIRT1 deacetylates WRN, decreasing its helicase and exo-
nuclease activities (193). Thus the interaction between
SIRT1 and WRN is another mechanism to regulate DNA
Nucleotide excision repair (NER) is a particularly impor-
tant mechanism by which the cell can prevent unwanted
mutations. The xeroderma pigmentosum C (XPC) protein
is essential for the initiation of the NER. SIRT1 enhances
XPC expression acting as a tumor suppressor through its
role in DNA repair (222).
4. FOXO1, -3, and -4
The Forkhead box, group O (FOXO) subfamily of fork-
head transcription factors is able to sense nutrient availabil-
ity and regulates various cellular processes, including apo-
ptosis and the cell cycle (2, 83). SIRT1 deacetylates the
FOXO proteins increasing their nuclear retention and tran-
scriptional activity (82, 175, 260). Activation of certain
members of the FOXO family can increase resistance to
oxidative stress (181), and SIRT1 is responsible for poten-
tiating this function (334). Specifically, SIRT1 regulates
FOXO3 by both inhibiting FOXO3-induced apoptosis and
and resist oxidative stress (54, 334). Other studies suggest
that SIRT1 modulation of FOXO1 is important for proper
glucose homeostasis, angiogenesis, and feeding behaviors
(96, 206, 281).
p300 is an acetyltransferase that regulates numerous signal-
ing pathways by facilitating transcriptional activity of a
broad array of transcription factors through modular sub-
domains. It was demonstrated that SIRT1 interacts with
and represses p300 transactivation in a NAD-dependent
manner (50). SIRT1 repression involves a transcriptional
repression domain of p300 named CRD1, which has two
residues (Lys-1020 and Lys-1024) that are essential for
lation (50). These residues also serve as acceptor lysines for
modification by the small ubiquitin-like modifier (SUMO)
protein. The SUMO-specific protease SSP3 relieves SIRT1
lase substrate for SIRT1 through a conserved SUMO con-
sensus motif (50).
Heat shock factor 1 (HSF1) increases transcription of heat
shock proteins in response to cell stress (142). These pro-
teins act to stabilize other proteins, thereby reducing pro-
tein misfolding. A recent study shows that SIRT1 deacety-
lates HSF1, increasing its binding activity with heat shock
promoter Hsp70 (350).
The peroxisome proliferator-activated receptor (PPAR) ?
belongs to the nuclear hormone receptor superfamily and
regulates gene expression upon heterodimerization with the
retinoid X receptor by ligating to peroxisome proliferator
response elements (PPREs) in the promoter region of target
genes. PPAR? is considered to be one of the master regula-
tors of adipocyte differentiation (329). It has been shown
that upon food withdrawal, SIRT1 stimulates fat mobiliza-
tion in white adipose tissue by repressing PPAR? (250). The
mechanism by which SIRT1 represses PPAR? involves the
cofactors NCoR (nuclear receptor corepressor) and SMRT
(silencing mediator of retinoid and thyroid hormone recep-
tors) (250). These effects are also observed in 3T3-L1 adi-
pocytes, where activation of SIRT1 inhibits adipogenesis,
while the inhibition of SIRT1 increases adipogenesis (250).
PGC-1? is the inducible coactivator-1? of PPAR?, which is
expressed at low levels in the liver during the fed state, but
at high levels after food deprivation (135). PGC-1? plays a
fatty acid oxidation. Consistent with this role, increased
expression of PGC-1? in the liver results in enhanced he-
patic glucose production (135). As discussed in more detail
below, SIRT1 deacetylates PGC-1? (275), and this appears
to be a crucial signal for the actions of SIRT1 at hepatic
PPAR? is a ligand-activated transcription factor that be-
longs to the steroid hormone receptor superfamily. PPAR?
is expressed predominantly in tissues that have a high level
of fatty acid catabolism, such as liver, heart, and muscle,
where it regulates the expression of a number of genes crit-
ical for lipid and lipoprotein metabolism. Hepatic deletion
of SIRT1 leads to impaired PPAR? signaling and decreases
fatty acid beta-oxidation, whereas overexpression of SIRT1
activates PPAR? (259). Importantly, the actions of SIRT1
on PPAR? require the deacetylation of PGC-1? (259).
The liver X receptors (LXRs) alpha and beta (LXR? and
LXR?) are nuclear receptors that act as cholesterol sensors.
SIRT1 deacetylates a single conserved lysine on each recep-
tor (K432 in LXR? and K433 in LXR?), inducing receptor
activity, which exerts important regulatory action on cho-
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lesterol homeostasis (194). Consistent with this SIRT1-me-
diated regulation, SIRT1 knockout (KO) mice have lower
levels of HDL cholesterol, while their low-density lipopro-
tein (LDL) cholesterol levels are unaffected (194). More-
over, SIRT1 KO mice show a reduced cholesterol transport
in macrophages and the liver, and this leads to an accumu-
lation of hepatic cholesterol (194).
Sterol regulatory element-binding protein (SREBP) 1c is a
transcriptional regulator of fatty acid synthesis enzymes. It
SREBP1c (256). The inhibition of hepatic SIRT1 activity
was associated with an increase in the acetylated active
nuclear form of SREBP-1c in the livers of ethanol-fed mice,
suggesting that the effect of ethanol on SREBP-1 is medi-
ated, at least in part, through SIRT1 inhibition (373).
Uncoupling protein 2 (UCP2) belongs to the family of mi-
given cell towards fatty acid fuel utilization (14). SIRT1
represses UCP2 transcription by binding directly to its pro-
moter (47). Consistent with this regulatory role, it was ob-
served that transgenic mice overexpressing SIRT1 in pan-
creatic ?-cells have lower levels of UCP2 and enhanced
insulin secretion (225). Additionally, the effects of SIRT1
inhibition in the brain decreasing food intake and promot-
ing synaptic changes are abrogated in mice deficient for
PTP1B is a member of the protein tyrosine phosphatase
family. PTP1B plays a crucial role as an insulin receptor
phosphatase, and PTP1B-deficient mice are more sensitive
to insulin, have improved glucose metabolism, and are re-
sistant to diet-induced obesity (94). It has been shown that
the beneficial effects of SIRT1 activation on insulin sensi-
tivity are, at least partially, mediated by the repression of
PTP1B transcription at the chromatin level (317). Resvera-
trol, which may be an activator of SIRT1, also suppresses
PTP1B, which might be a key step for enhancing insulin
The transcription factors of the MyoD family have essential
functions in myogenic lineage determination and skeletal
muscle development. These myogenic regulatory factors ac-
tivate muscle-specific transcription of numerous genes.
SIRT1 has been shown to retard muscle differentiation
through its interaction with MyoD (111). However, SIRT1
does not interact directly with MyoD, but forms a complex
with the acetyltransferase p300/CBP-associated factor
(PCAF), and then deacetylates both PCAF and MyoD
The histone acetyltransferase TIP60 can acetylate many
substrates, including histones and p53, promoting apopto-
sis. SIRT1 deacetylates and inactivates TIP60 activity in
vivo, suggesting that SIRT1 plays an important role in the
control of histone acetyltransferase activity and function in
response to DNA damage (346).
regulates diverse biological processes including immunity,
inflammation, and apoptosis. SIRT1 physically interacts
with the RelA/p65 subunit of NF-?B and inhibits transcrip-
tion by deacetylating RelA/p65 at lysine 310 (369). The
interaction between SIRT1 and the NF-?B signaling path-
way is also involved in the cigarette smoke-mediated pro-
inflammatory cytokine release (364). This interaction was
observed in a monocyte-macrophage cell line and in inflam-
matory cells of rat lungs. However, one important role of
NF-?B is to mediate pancreatic ?-cell damage. Studies in
isolated rat islets or RINm5F cells have demonstrated that
overexpression of SIRT1 completely blocked cytokine-in-
duced cell damage (192), indicating that the SIRT1/NF-?B
pathway plays an important role mediating ?-cell damage.
Overexpression of SIRT1 was further shown to attenuate
hepatic NF-?B activation in vitro and in vivo (249).
Studies performed in vitro have shown that resveratrol in-
hibited the phosphorylation of insulin receptor substrate 1
(IRS1) Ser-307 and IRS2 Thr-348, which are markers of
insulin resistance (92), indicating that resveratrol improves
insulin signaling (106). In agreement with this, SIRT1 over-
expression increased the phosphorylation of PKB, a down-
stream target of insulin signaling, in muscle cells and
HEK293 cells, whereas the inhibition of SIRT1 induced the
opposite response (106). Furthermore, suppression of
SIRT1 activity inhibited the tyrosine phosphorylation of
insulin receptor substrate 2 (IRS2) in HEK293 cells (380).
In neurons, the inhibition of SIRT1 increased the acetyla-
tion and reduced the phosphorylation of IRS2 (196). The
reduced activity of SIRT1 also reduced Ras activation and
ERK1/2 phosphorylation (both markers of oxidized pro-
teins and lipids) (196). Thus this article suggests that SIRT1
can protect neurons from oxidative damage.
E2F1 is known to induce the transcription of several apo-
ptotic genes and can induce apoptosis after DNA damage
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events through both p53-dependent and p53-independent
mechanisms. SIRT1 inhibits E2F1 (343), and this inhibition
suppresses p73 transcriptional activity (81, 247). It seems
that SIRT1 interacts with PCAF and E2F1 on the P1p73
promotor (247), and thereby can modulate tumor prolifer-
MEF2 is a family of transcription factors with important
functions in muscle cell differentiation and apoptosis (216).
SIRT1 can potently induce MEF2 deacetylation during
muscle cell differentiation (384), indicating that this mech-
anism might be responsible of the negative regulation of
myogenesis by SIRT1.
The CREB regulated transcription coactivator 2 (CRTC2
or TORC2) plays an essential role in gluconeogenesis. For
instance, the stimulation of the gluconeogenic pathway in-
duced by glucagon requires the dephosphorylation and nu-
clear translocation of TORC2. SIRT1 reduces TORC2 ac-
tivity in the liver, while the inhibition of SIRT1 signaling
induces TORC2 activity (206), suggesting that this path-
way is an important modulator of the gluconeogenesis (dis-
Sirtuins and PARPs act as survival and death inducing fac-
tors. These two protein families are both dependent on
NAD?for their activities. In response to DNA damage,
it protects cells from PARP1-mediated cell death (178,
Hypoxia-inducible factor 1? (HIF1?) is a transcription fac-
tor that under hypoxic conditions regulates the transcrip-
tion of hundreds of genes in a cell type-specific manner
(297). SIRT1 binds and deacetylates HIF1?, and therefore
inactivates HIF1? by blocking p300 recruitment (199). In
hypoxic conditions, SIRT1 is downregulated due to de-
creased NAD?levels, which allows the acetylation and ac-
tivation of HIF1? (199).
B. Mitochondrial Targets of SIRT3
The NAD?-dependent deacetylase SIRT3 is localized in mi-
tochondria (207, 218, 236, 290, 294, 300) and plays an
important role in mitochondrial metabolism (300). To ex-
ert these metabolic actions, SIRT3 regulates the expression
of many mitochondrial proteins through a reversible
deacetylation process (125, 293). Some of the SIRT3 sub-
strates discussed below are nuclear, and given the fact that
necessary to clarify the interaction between SIRT3 and nu-
clear proteins (FIGURE 5).
Acetyl-CoA synthase 2 (AceCS2) was the first SIRT3 mito-
chondrial protein substrate to be identified (125, 293) and
is an enzyme that catalyzes the production of acetyl-CoA
ible acetylation; SIRT3 deacetylates and activates AceCS2,
whereas acetylation of AceCS2 inhibits its activity (125,
293). Since Acetyl-CoA is an important regulator of several
metabolic pathways, including cholesterol and fatty acid
biosynthesis, it is possible that SIRT3 modulates those bio-
Hydroxyl methylglutaryl CoA synthase 2 (HMGCS2), the
rate-limiting step in ?-hydroxybutyrate synthesis, was
shown to be deacetylated and activated by SIRT3 in the mito-
chondria (301). Upon fasting, SIRT3-mediated HMGCS2 acti-
vation was required to induce the production of ketone
Glutamate dehydrogenase (GDH) is a key metabolic en-
zyme that is colocalized with SIRT3 in the mitochondrial
matrix (290). SIRT3 deacetylates and activates GDH in
vitro (290) and in vivo (207).
SIRT3 suppresses p53 activity leading to growth arrest and
senescence (363). This was demonstrated in the EJ-p53 cell
line, where SIRT3 colocalizes with p53 in the mitochondria
before inducing cell arrest (363). A close interaction be-
tween SIRT3 and p53 also plays an important function in
mouse preimplantation in vitro (167), even though mice
lacking SIRT3 are fertile.
SIRT3 protects cardiomyocytes from stress-induced cell
death by deacetylating Ku70, leading to the interaction be-
tween Ku70 and the proapoptotic protein Bax. This path-
way is associated with a protective function of SIRT3 on
cardiomyocytes under stress conditions (318, 319).
SIRT3 activates FOXO3a in cardiomyocytes leading to the
activation of a molecular pathway involving ROS/Ras/
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MAPK/ERK and PI3K/Akt, which ultimately inhibits car-
ocytes have been also demonstrated in vivo, as mice lacking
SIRT3 shows signs of cardiac hypertrophy at 8 wk of age
(318). Similar results were obtained in C. elegans, where
daf-16, the homolog of the human FOXO family, was also
deacetylated by SIRT3 (155).
Acetylation of histone H3 core domain lysine 56 (H3-K56)
is essential for the compaction of DNA into chromatin, and
the histone acetyltransferase p300 acetylates H3-K56 in
vivo. Human SIRT3 deacetylates H3K-56 (341), indicating
that SIRT3 regulates the DNA damage response pathway.
As previously mentioned, the nonmitochondrial localiza-
tion of SIRT3 remains to be fully confirmed. As such, the
8. Cyclophilin D
Increasing evidence implicates a multi-protein complex
called the mitochondrial permeability transition pore
(mPTP) in the decline in mitochondrial function with age.
low-level, chronic triggering results in mitochondrial swell-
ing, membrane depolarization, and the destruction of de-
fective mitochondria by autophagy. SIRT3 deacetylates the
regulatory component of the mPTP, cyclophilin D (CypD)
a CypD inhibitor (122, 304). Consistent with this, cardiac
myocytes from mice lacking SIRT3 exhibit an age-depen-
dent increase in mitochondrial swelling due to increased
mPTP opening (122). SIRT3?/?mice show accelerated
signs of aging in the heart including cardiac hypertrophy
and fibrosis at 13 mo of age and are hypersensitive to heart
stress. Deacetylation of cyclophilin D also induces dissoci-
ation of hexokinase II from the mitochondria and prevents
on glycolysis to oxidative phosphorylation (304).
The mitochondrial ribosomal protein L10 (MRPL10) plays
an important function in the acetylation in the mitochon-
drial chromosome. SIRT3 overexpression in C2C12 cells
induces the deacetylation of MRPL10 and thereby dimin-
n of cardiomyocytes
Regeneration of antioxidants
and citric acid cycle
fatty acid biosynthesis
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ishes the synthesis of proteins in the mitochondria (367).
Consistent with these findings, the inhibition of SIRT3 in
those cells leads to an increased synthesis of mitochondrial
Succinate dehydrogenase (SDH) is a unique enzyme, as it par-
ticipates in both the tricarboxylic acid cycle and oxidative
phosphorylation in mitochondria. Therefore, it is essential for
subunits, SDhA, is a substrate for SIRT3, allowing this sirtuin
to regulate oxidative phosphorylation (59).
SIRT3 was shown to deacetylate and thereby activate orni-
thine transcarbamoylase (OTC), a rate-limiting enzyme in
the urea cycle (126). Calorie restriction increased both
SIRT3 and OTC activity; in SIRT3 null mice, low OTC
activity during fasting was connected with increased orotic
acid levels, indicating a disturbed nitrogen balance. Hallow
drial proteins in SIRT3 null and wild-type mice and re-
vealed a number of novel SIRT3 targets involved in ?-oxi-
dation and amino acid catabolism.
The isocitrate dehydrogenase 2 (ICDH2) is another key met-
abolic regulator in the mitochondrial matrix that is also colo-
calized with SIRT3 in the mitochondrial matrix. ICDH2 is
also deacetylated and activated by SIRT3 (290), an effect that
was abolished by a sirtuin inhibitor. The precise acetylation
sites for ICDH2 (K75 and K241) were found to be deacety-
lated by SIRT3 (290). The increased activity of ICDH2 pro-
motes regeneration of antioxidants and catalyzes the citric
SIRT3 was shown to augment the mitochondrial glutathione
antioxidant defense system in mice via increasing NADPH
levels and the ratio of reduced-to-oxidized glutathione in mi-
tochondria (310). Importantly, calorie restriction-induced
SIRT3 activation was thereby linked to the prevention from
extensive age-related oxidative damage (310).
Recently, SIRT3 was shown to deacetylate manganese su-
peroxide dismutase (MnSOD), thereby increasing MnSOD
activity (318, 325). Such SIRT3-mediated MnSOD activa-
tion consequently decreased mitochondrial superoxide.
Conversely, SIRT3 ablation in mice decreased hepatic Mn-
SOD activity, which might contribute to the tumor-permis-
sive environment seen in SIRT3?/?animals. Accordingly,
SIRT3?/?mice displayed higher susceptibility to radiation-
induced liver damage (325). On the other hand, SIRT3
blocked cardiac hypertrophy via MnSOD activation, sug-
gesting that SIRT3 is an important negative regulator of
cardiac hypertrophy (318).
14. ATP and the electron transport chain
A recent study shows that SIRT3 deacetylates one or more
proteins of the electron transport chain complex I, includ-
ing NDUFA9 (5). Complex I activity is inhibited in
SIRT3?/?mice and potentiated in mitochondria that have
been exposed to increased levels of SIRT3 (5). These results
suggest that SIRT3 plays an important role in regulating
ATP synthesis in mitochondria and thus is a potential reg-
ulator of mitochondrial energy metabolism (5).
V. ENDOGENOUS REGULATORS OF SIRT1
Besides the classical activation of sirtuins by increased
NAD?/NADH ratios, a number of other endogenous pro-
cesses regulate SIRT1 and SIRT3 activity. Transcription of
sirtuins can be controlled by a number of transcription fac-
tors and transcriptional coactivators or repressors. Further-
more, mRNA stability of sirtuins depends on the presence
of specific microRNAs. Last, sirtuin activity can be regu-
lated by posttranslational modifications through protein-
protein interaction, stimulating or repressing sirtuin activ-
ity (FIGURES 4 and 5).
A. Endogenous Regulators of SIRT1
E2F1 induces SIRT1 expression at the transcriptional level
(53, 343). E2F1 is also a substrate of SIRT1, and deacety-
lation of E2F1 inhibits its activity as a transcriptional acti-
vator. This action is modulated by the interaction of SIRT1
with PCAF, which controls the E2F1/p73 apoptotic path-
way (247). Accordingly, the downregulation of SIRT1 in-
creases E2F1 transcriptional and apoptotic functions (53,
343). Therefore, this SIRT1-E2F1 negative-feedback loop
might act as a regulatory switch that can determine the
apoptotic fate of a cell.
In addition to being a target for SIRT1 deacetylation, p53
can also repress SIRT1 transcription through binding to
two response elements within the SIRT1 promoter. p53-
null mice have increased levels of SIRT1 in different tissues,
and several p53-null tumor cell lines display SIRT1 overex-
pression (104, 229). Therefore, SIRT1 and p53 also form a
regulatory feedback loop since SIRT1 is known to deacety-
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The growth regulator and tumor repressor gene Hyperm-
ethylated In Cancer 1 (HIC1) also suppresses SIRT1 tran-
scription. HIC1, COOH-terminal binding protein 1
(CTBP1), and SIRT1 form a corepressor complex (76) that
binds enhancer elements upstream of the SIRT1 promoter
and inhibits SIRT1 expression. In both mouse and human
prostate cancer cells, as well as Hic?/?mouse embryonic
an increase in SIRT1 expression levels (143), indicating one
HuR is a ubiquitously expressed RNA-binding protein that
stabilizes mRNAs and regulates translation to protein. The
tumor suppressor HuR (also known as ELAVL1) is an
mRNA binding protein that binds the 3= UTR of SIRT1
mRNA, stabilizes the SIRT1 mRNA, and increases SIRT1
expression levels (1). Moreover, HUR gene expression cor-
relates with the reduced levels of SIRT1 expression in senes-
cent cells (1).
MicroRNAs are posttranscriptional gene regulators that
are differentially expressed under several physiological con-
ditions. The microRNA miR-34a also binds the 3= UTR of
SIRT1 mRNA (358, 359). In contrast to HUR, miR-34a
prevents translation of SIRT1 and so inhibits SIRT1
deacetylase activity, subsequently inducing the accumula-
tion of acetylated p53.
In addition to miR-34a, miR-199a has also been associated
with SIRT1 regulation. Knockdown of miR-199a results in
the upregulation of SIRT1 and HIF1? (271). As this report
found that miR-199a levels were decreased in cardiac myo-
cytes upon hypoxia, the data indicate that SIRT1 is impor-
tant for hypoxic damage (271).
Deleted in breast cancer 1 (DBC1, also known as
KIAA1967) is an inhibitor of SIRT1 activity (170, 172,
382). Moreover, reduction of DBC1 inhibits p53-mediated
apoptosis after induction of double-stranded DNA breaks
owing to SIRT1-mediated p53 deacetylation. Although lit-
tle is known about the normal function of DBC1, its loss in
several cancer cell lines and its inhibition of SIRT1 suggest
it may have an important role in tumorigenesis.
Active regulator of SIRT1 (AROS, also known as RPS19BP1)
is a 142-amino acid nuclear protein, which directly activates
SIRT1 activity and attenuates p53-dependent transcriptional
increased cell susceptibility to apoptosis after DNA damage
Necdin is a member of the melanoma-associated antigen
(MAGE) family of proteins. These proteins play multiple
functions in cellular regulation, including tumorigenesis or
neuronal differentiation and survival. Necdin has been
shown to negatively regulate p53 by potentiating SIRT1-
ylation through a necdin-SIRT1-p53 complex prevents
p53-mediated apoptosis in response to DNA damage.
AMPK controls the expression of genes involved in energy
metabolism in mouse skeletal muscle by acting in coordina-
tion with SIRT1. AMPK increases SIRT1 activity by in-
creasing cellular NAD?levels (57). Interestingly, this inter-
SIRT1 activation stimulates fatty acid oxidation and indi-
rectly activates AMPK (99).
Forkhead box O-class (FOXO) transcription factors func-
tion as tumor-suppressor proteins by inhibiting cell prolif-
eration, promoting apoptotic cell death, and protecting
cells from DNA damage and oxidative stress. FOXO3a
stimulates SIRT1 transcription through two p53 binding
sites present in the SIRT1 promoter (229). Consistently,
knockdown of FOXO3a expression inhibits the starvation-
induced increase in SIRT1 expression (229).
c-Myc regulates processes involved in many, if not all, aspects
of cell fate. c-Myc and SIRT1 form a negative-feedback loop
that inhibits c-Myc-induced cellular transformation. On one
lates c-Myc, resulting in decreased c-Myc stability and subse-
quent suppression of SIRT1 expression (375).
Orphan nuclear receptor small heterodimer partner (SHP)
is a transcriptional corepressor of a wide variety of nuclear
receptors. It has been reported that SHP interacts with and
colocalizes with SIRT1, inhibiting the transcriptional activ-
ity of SIRT1 (71).
14. PARP1 and PARP2
The deletion of PARP1, a gene encoding a major NAD?-con-
suming enzyme, increases SIRT1 activity in brown adipose
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tissue and muscle. Also, the pharmacological inhibition of
PARP in vitro and in vivo increased SIRT1 activity (24). Sim-
ity in cultured myotubes (23). However, unlike PARP1,
PARP2 acts as a negative regulator of the SIRT1 promoter,
and this regulation is independent of NAD?levels.
B. Endogenous Regulators of SIRT3
report suggested that PGC-1? stimulates mouse SIRT3 ac-
tivity in both muscle cells and hepatocytes (180). In agree-
ment with this, SIRT3 knockdown suppressed the actions
of PGC-1? on mitochondrial biogenesis in myotubes (180).
VI. METABOLIC TISSUES TARGETED
A. Roles of SIRT1 in Liver Metabolism
1. SIRT1 and hepatic glucose metabolism
In liver, SIRT1 is upregulated during negative energy bal-
ance, as occurs during fasting and calorie restriction (7, 61,
229, 233, 275). In 2005, research led by Puigserver and
collaborators (275) revealed that during fasting SIRT1
stimulates gluconeogenesis and inhibits glycolysis by
deacetylating the transcriptional coactivator PGC-1?. The
authors showed that SIRT1 interacts with and deacetylates
PGC-1? in a NAD?-dependent manner (230, 275). Impor-
tantly, the regulation of PGC-1? by SIRT1 was found to be
essential for fasting- or pyruvate-mediated increases of glu-
coneogenic genes (PEPCK and G6Pase) and glucose output
in cultured hepatocytes. Furthermore, this deacetylation
also acts to decrease the expression of glycolytic genes (glu-
cokinase and LPK) (275). This effect of SIRT1 stimulating
mice (96, 274).
SIRT1 has been shown to regulate hepatic glucose metabo-
lism (FIGURE 6) by interacting with the FOXO family of
transcription factors (166, 177). FOXO proteins are inac-
tivated by the PI3k-Akt/PKB pathway in response to hor-
monal signaling (32, 120). When phosphorylated, these
transcription factors are inactive and are localized in the
cytosol; upon dephosphorylation, they have nuclear local-
ization and are active (383). Several groups have shown the
close and intricate interaction between SIRT1 and FOXO.
Motta et al. (224) found that SIRT1 binds to FOXO3a and
promotes its deacetylation with consequent inhibition of
FOXO3a transcriptional activity. On the other hand,
FOXO3a induced by starvation promotes SIRT1 expres-
was also shown to interact and deacetylate FOXO1 (365)
and FOXO4 (177). The FOXO family of transcription fac-
tors has been shown to stimulate the expression of genes
involved in gluconeogenesis (pepck and g6pase) (353, 368),
while decreasing expression of glucose kinase (gk) (21, 292,
381). During prolonged fasting, FOXO1 activity supported
Fatty acid oxidation
Metabolic roles of SIRT1 in peripheral tissues and the central nervous system.
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by SIRT1 seems to be a key element in the maintenance of
to stimulate FOXO1 translocation to hepatocyte nuclei
(105). Under conditions of SIRT1 activation or oxidative
stress, FOXO1 is confined to a nuclear subdomain where it
boosts transcription of genes that promote gluconeogenesis
and hepatic glucose production (105). Importantly, muta-
tions of the FOXO1 coactivator-interacting LXXLL motif
eliminated FOXO1 interaction with SIRT1, indicating that
this motif is essential for SIRT1 actions mediated by
SIRT1 is also known to regulate gluconeogenesis via its
interaction with STAT3 (233). STAT3 suppresses gluco-
neogenesis by inhibiting the transcription of PEPCK1 and
lysine sites that are acetylated. This acetylation has been
shown to regulate the activity and phosphorylation of this
protein. SIRT1 deacetylases STAT3, thus decreasing
STAT3 activity and its inhibitory effect on gluconeogenesis.
Therefore, activation of SIRT1 during fasting stimulates
gluconeogenesis by inhibiting STAT3 while activating
PGC-1? and FOXO1.
In contrast, a number of studies suggest that the activation
of SIRT1 inhibits insulin-induced hepatic glucose produc-
tion in obese rats (220). Moreover, a recently identified
fasting-inducible switch, consisting of SIRT1 and p300,
causes a transfer between two key regulators of glucose
production (206). The switch occurs during nutrient depri-
vation, when regulation of the gluconeogenic program is
shifted from CREB-regulated transcription coactivator 2
ing, glucagon induces p300/CBP-mediated acetylation of
CRTC2, which briefly increases gluconeogenic activity.
During prolonged fasting, SIRT1 deacetylates and down-
regulates CRTC2 and promotes a FOXO1-mediated gluco-
neogenic program (206). Therefore, SIRT1 may also act to
suppress hepatic glucose production during prolonged fast-
2. SIRT1 and hepatic fatty acid metabolism
SIRT1 plays a prominent role in the regulation of hepatic
fatty acid metabolism (FIGURE 6). Specifically, SIRT1 reg-
ulated hepatic lipid metabolism by activating the AMPK/
LKB1 signaling pathway (140). Notably, LKB1 was es-
sential to produce downstream effects of SIRT1 on fatty
acid oxidation and lipogenesis (140). In a hyperglycemic
environment, SIRT1-mediated activation of AMPK pre-
vents lipid accumulation and FAS induction (140). Con-
sistent with this regulation, mice fed a high-fat diet
treated with resveratrol or the SIRT1 agonist SRT1720
exhibited improved liver physiology and metabolic func-
tion (34, 220, 249).
A subsequent study suggests that although SIRT1 improves
insulin sensitivity, transgenic mice overexpressing SIRT1
on an atherogenic diet display elevated hepatic lipid accu-
mulation and secretion, in spite of enhanced glucose ho-
3. SIRT1 and hepatic cholesterol metabolism
The cholesterol-sensing LXR proteins are nuclear receptors
involved in maintaining cholesterol homeostasis. LXRs act to
enhance the reverse transport of cholesterol from peripheral
tissues by stimulating the expression of the ATP-binding cas-
teins (194). SIRT1 interacts with and deacetylates the LXRs,
influencing several targets including the ABCA1 (194), and
therefore, regulating HDL production (19). SIRT1 also mod-
ulates cholesterol metabolism through the critical regulator
SREBP. In cultured cells, the activation of SIRT1 increases
SREBP ubiquitination, decreases SREBP nuclear levels, and
diminishes the pool of active SREBP, thereby decreasing
SREBP gene expression (342). SIRT1 seems to have a con-
served role in metazoans in the repression of SREBP during
(342). In vivo, the administration of the synthetic SIRT1 acti-
vator SRT1720 to leptin-deficient mice fed a high-fat diet in-
hibited the expression of SREBP and improved the hepatoste-
B. Roles of SIRT1 in Pancreatic ?-Cells
1. SIRT1 and insulin secretion
In addition to the regulation of hepatic glucose metabolism,
SIRT1 has also been linked to glucose-stimulated insulin
secretion in pancreatic ?-cells (47, 225) (FIGURE 6). A selec-
tive increase in the dosage of SIRT1 in pancreatic ?-cells
improves glucose tolerance and insulin release in response
to glucose and KCl (225). Conversely, SIRT1 knockdown
in ?-cells had the opposite effect on insulin secretion (47).
This effect appears to be partially induced via SIRT1-medi-
ated repression of ucp2 (47). Intriguingly, this improved
insulin sensitivity is not exclusive to the ?-cell. SIRT1 was
shown to improve insulin sensitivity by decreasing the tran-
a negative regulator of insulin receptor signaling, and its
downregulation improves insulin sensitivity (94). Trans-
genic mice with mild overexpression of SIRT1 also showed
improved glucose tolerance in several models of insulin re-
sistance, an effect that was mediated by decreased hepatic
production of glucose and increased adiponectin levels, and
not by differences in ?-cell sensing of glucose (28). Alterna-
tively, SIRT1 may act as a mediator for other signals. For
instance, wallerian degeneration slow, a fusion protein that
is highly expressed in the pancreas and improves glucose
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homeostasis, increases SIRT1 activity to downregulate
UCP2 expression, leading to an improvement in glucose
homeostasis (355). On the other hand, glucagon-like pep-
tide 1 (GLP-1) inhibits SIRT1. This inhibition is essential
for the GLP-1 stimulation of ?-cell mass expansion (33).
2. SIRT1 and ?-cell protection
SIRT1 also plays a role in pancreatic beta cell protection. In
the hyperglycemic state, oxidative stress can induce pancre-
atic beta cell failure (175). SIRT1, FOXO1, and Pml (pro-
myelocytic leukemia protein) form a complex that upregu-
lates transcription factors (NeuroD and MafA) to protect
?-cells from hyperglycemia-induced damage (175). Also,
the SIRT1-mediated regulation of NF-?B may prevent pan-
creatic ?-cell toxicity (192). SIRT1 has been shown to reg-
ulate NF-?B by deacetylating lysine 310 on its relA/p65
C. Roles of SIRT1 in Skeletal Muscle
1. SIRT1 and skeletal muscle fatty acid metabolism
When skeletal muscle is nutrient-deprived, glucose oxida-
tion shifts to fatty acid oxidation. SIRT1 is activated during
low energy states, and therefore, it makes sense that SIRT1
would induce fatty acid oxidation as the cell shifts from
glucose consumption to fatty acid oxidation (114) (FIGURE
mitochondrial fatty acid oxidation in a glucose-scarce envi-
ronment (114). SIRT1 deacetylates and activates acetyl-
CoA synthetase (AceCS) 1, which can induce substantial
fatty acid synthesis (125). AceCS1 is regulated by reversible
acetylation, a process in which acetylation inactivates
AceCS1 and SIRT1-induced deacetylation reactivates it. In
SIRT1 activation is critically discussed in section X) signif-
icantly increased their aerobic capacity (186). The effects of
resveratrol were also associated with an induction of genes
for oxidative phosphorylation and mitochondrial biogene-
sis. Furthermore, these effects were largely explained by the
deacetylation of PGC-1? and subsequent increases in
PGC-1? activity (186). Importantly, resveratrol protected
these mice against diet-induced obesity and insulin resis-
tance (186). SIRT1 may also act on uncoupling protein 3
(UCP3), a mitochondrial protein expressed in skeletal mus-
cle that lowers membrane potential and prevents fatty acid
accumulation (11). This regulation occurs via SIRT1-medi-
ated deacetylation of histones near the UCP3 promoter,
thereby preventing UCP3 transcription and subsequently
the accumulation of fatty acids (11).
2. SIRT1 and skeletal muscle glucose metabolism
As discussed in section IVA13, in vitro studies demon-
strated that SIRT1 improves insulin sensitivity by repress-
ing the protein tyrosine phosphatase 1B (PTP1B) gene in
be more insulin sensitive and more resistant to diet-induced
obesity compared with controls (94). In vivo studies have
also shown that resveratrol was able to increase glucose
uptake, an effect independent of insulin and dependent on
AMPK, through the increase of the intrinsic activity of the
glucose transporter GLUT4 (52). Further in vivo study has
demonstrated an increased activity of SIRT1 in muscle
when mice were under caloric restriction, an effect that was
completely abrogated in mice lacking SIRT1 (288). The
mechanisms for this activation likely involve the Stat3/PI3K
pathway as SIRT1 is known to inactivate the transcription
factor Stat3 during caloric restriction, resulting in more
efficient PI3K signaling during insulin stimulation (288).
3. SIRT1 and skeletal muscle differentiation
SIRT1 was shown to negatively regulate muscle gene ex-
pression and differentiation. To exert these actions, SIRT1
does not directly interact with muscle transcriptional regu-
lators, but rather associates with the complex PCAF/
GCN5. In the presence of PCAF/GCN5, SIRT1 associates
with and deacetylates both transcriptional factors PCAF
and myogenic determining factor (MyoD) (111). More-
over, in C2C12 myotubes, when MyoD is present with
SIRT1 on the PGC-1? promoter, PGC-1? overexpression
increases due to positive feedback of PGC-1? on its own
promoter (10). Therefore, MyoD enhances the overexpres-
sion of PGC-1? in a SIRT1-dependent manner (10).
D. Roles of SIRT1 in White Adipose Tissue
1. SIRT1 and adipose tissue fatty acid metabolism
SIRT1 favors lipolysis and fatty acid mobilization in response
to fasting by repressing PPAR? (250), which is essential for
adipogenesis (FIGURE 6). SIRT1 interacts with PPAR? DNA
binding sites, but it is unclear whether SIRT1 deacetylates
PPAR? directly (250). Another pathway modulating the lipo-
of FOXO1 and stimulation of adipose triglyceride lipase
(ATGL) gene transcription (69). Activation of SIRT1 inhibits
inhibition of SIRT1 activates adipocyte differentiation sub-
stantially (22). In pig preadipocytes, SIRT1 may inhibit
ment (22). SIRT1 has been shown to regulate adipose tissue
inflammation by suppressing induction of proinflammatory
transcription in response to fatty acids, hypoxia, and endo-
plasmic reticulum stress (79, 115). For instance, the reduction
of SIRT1 in vivo stimulates macrophage recruitment to adi-
pose tissue, whereas overexpression of SIRT1 prevents adi-
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pose tissue macrophage accumulation during chronic high-fat
2. SIRT1 and adipose tissue glucose metabolism
SIRT1 also regulates the production and/or the secretion of
insulin-sensitizing factors such as adiponectin and FGF21
through the regulation of FOXO1 and PPAR? (28, 262,
345). These studies suggest that in adipocytes, PPAR? se-
lectively represses the expression of a group of genes con-
taining FGF21. However, in vivo studies have yet to cor-
roborate these findings. Moreover, the adipocytes of mice
sity and type 2 diabetes, show lower levels of FOXO1 and
SIRT1 than their littermates (315), whereas the activation
of SIRT1 by resveratrol rescued FOXO1 levels in the adi-
pocytes of db/db mice (315). The beneficial role of SIRT1
on glucose metabolism might be at least partially explained
by the activation of adiponectin. Adiponectin is an impor-
tant regulator of energy homeostasis, and its expression is
reduced in obesity and type II diabetes. SIRT1 activates
FOXO1, which forms a transcription complex with
CCAAT/enhancer-binding protein ? (C/EBP?), thus pro-
moting adiponectin gene transcription (262). SIRT1 may
also play a role in the attenuation of ROS. In adipocytes
exposed to high levels of free fatty acids, activation of
SIRT1 leads to nuclear translocation of FOXO1 and is ac-
companied by a parallel decrease in ROS production (315).
SIRT1-mediated activation of FOXO1 may therefore pro-
tect from obesity-induced inflammation and subsequently
E. Central Nervous System SIRT1
Regulates Metabolic Control
SIRT1 is present in metabolically important areas of the
brain, including the arcuate, ventromedial, dorsomedial,
and paraventricular nuclei of the hypothalamus, as well as
the area postrema and the nucleus of the solitary tract in the
hindbrain (268) (FIGURE 6). SIRT1 protein levels are mod-
ulated by nutrient status, since fasting increased the hypo-
hypothalamus of leptin-deficient mice (268). Specifically,
SIRT1 mRNA is expressed in pro-opiomelanocortin neu-
rons, which are essential integrators of proper glucose ho-
meostasis and body weight regulation (268). Several studies
suggest that SIRT1 regulates food intake and body weight
through the central melanocortin system of the brain (55,
87, 281). For instance, a decrease in hypothalamic SIRT1
signaling by pharmacological inhibition or by siRNA pre-
and body weight gain (55, 87, 340). Central administration
of an orexigenic melanocortin receptor antagonist reversed
the effects of SIRT1 inhibition (55, 87), supporting the hy-
pothesis that hypothalamic SIRT1 interacts with the central
melanocortin system in a FOXO1-dependent manner.
Pharmacological inhibition of brain SIRT1 decreased the
activity of AgRP neurons and the inhibitory tone of POMC
neurons (87). This inhibition of SIRT1 decreased food in-
take through the central melanocortin receptors in a UCP2-
ade of SIRT1 in the central nervous system prevents the
pharmacological results, the selective disruption of SIRT1
food intake (87). Also, the AgRP-SIRT1 KO mice displayed
decreased fat mass and impaired metabolic response to fast
(87). During fasting, increased SIRT1 expression represses
FOXO1 activity through increased deacetylation (55). In-
creased SIRT1 expression also appears to activate S6K sig-
naling (55). Consistently, the overexpression of SIRT1 in
the mediobasal hypothalamus rescues hyperphagia and
body weight gain induced by constitutive nuclear FOXO1
expression and thereby suppresses the expression of the
orexigenic neuropeptide AgRP (281).
There are controversial data surrounding intracerebroven-
tricular (ICV) infusion of the proposed SIRT1 activator
resveratrol. Chronic ICV infusion of resveratrol signifi-
cantly decreased insulin resistance and normalized blood
Interestingly, despite diminished I?-B? mRNA levels after
ICV resveratrol infusion, hypothalamic NF-?B signaling
was augmented via deacetylation of RelA/p65 and total
RelA/p65 protein (267). Another study using resveratrol
has shown that the acute central injections of this com-
pound in the mediobasal hypothalamus during basal
pancreatic insulin clamp studies improved glucose ho-
meostasis through a central SIRT1-dependent pathway
(176). The mechanism(s) behind these controversial ef-
fects has yet to be determined, but it is important to note
that several studies suggest that resveratrol is not an ac-
tivator of SIRT1, and therefore, the effects observed after
central administration of resveratrol are not exerted
through hypothalamic SIRT1.
Additional studies have reported the effect of the lack of
lacking SIRT1 in the brain showed defects in somatotropic
specifically, brain SIRT1 specific KO mice are dwarfed and
old mice are glucose intolerant and display altered caloric
restriction responses (60). Another report investigating the
heavier than their littermates and had more perigonadal fat
due to lower energy expenditure (266). The deletion of
SIRT1 in POMC neurons also abolished the capacity of
leptin to activate phosphoinositol 3-kinase (PI3K) signaling
in POMC neurons and altered brown adipose tissue and
perigonadal white adipose tissue metabolism (266).
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In addition to genetic disruption of SIRT1 signaling in the
central nervous system, the effect of overexpression of
SIRT1 in the brain has been also studied. Elevated levels of
SIRT1 in hypothalamic dorsomedial and lateral nucleus
increased body temperature and physical activity in re-
sponse to caloric restriction (282). In summary, central
SIRT1 signaling seems to be a key mediator of energy me-
tabolism and the physiological response to calorie restric-
tion. However, the exact mechanisms of central SIRT1 ac-
tion remain to be elucidated.
F. Roles of SIRT1 in Other Tissues Involved
in Metabolic Control
SIRT1 is expressed in a number of additional tissues that
are important for metabolic control. For instance, SIRT1 is
abundantly expressed in the mouse kidney, particularly in
renal medullary interstitial cells (131). Studies in cultured
renal medullary interstitial cells have shown that SIRT1
protects from oxidative injury (131) and hypoxia (182), via
this observation, SIRT1 is downregulated during condi-
tions of hypoxia, and therefore activates HIF1? (199).
SIRT1 is further expressed in a number of myeloid cells,
where it can exert metabolic control. For instance, macro-
phage SIRT1 was shown to improve insulin sensitivity by at-
tenuating inflammatory pathways (372). To date, nothing is
known on the role of SIRT1 in metabolically relevant tissues
known to suppress intestinal tumorigenesis and colon cancer
growth (102). However, nothing is known yet on potential
roles of intestinal SIRT1 in metabolic control.
VII. METABOLIC TISSUES TARGETED BY
A. Roles of SIRT3 in Liver Metabolism
Gene and protein expression of SIRT3 in the liver were
activated by fasting (136) and decreased when the animals
were fed a high-fat diet (HFD) (30, 168). HFD feeding in
SIRT3 KO mice induced an increase in the acetylation of
several hepatic proteins, suggesting that SIRT3 is a crucial
regulator of hepatic mitochondrial function (168). Mecha-
consumption and ROS levels in hepatocytes in a SIRT3-
dependent manner (30). Palmitate increases oxygen con-
sumption in hepatocytes, and the inhibition of SIRT3 abol-
ished those actions (30). Similar results were obtained in
mice lacking SIRT3, as a palmitate test in these mice in-
duced lipotoxicity in hepatocytes, whereas this effect was
abrogatedwhenSIRT3levelswererestored(30) (FIGURE 7).
The relevance of SIRT3 in hepatic metabolism was also
confirmed in another study showing that SIRT3 overex-
(299). This effect appeared to be mediated by the activation
of AMPK and ACC (299); when AMPK was inhibited by
compound C, SIRT3 failed to decrease lipid accumulation
(299). In this line, another study demonstrated that SIRT3
induced fatty acid oxidation through the deacetylation of
long-chain acyl coenzyme A dehydrogenase (LCAD) and
mice lacking SIRT3 showed lower fatty acid oxidation
(136). Very recently, it was suggested that hepatic ketone
body production is regulated by SIRT3; HMGCS2, the rate
limiting enzyme in the synthesis of ?-hydroxybutyrate,
needs deacetylation to induce the physiological response to
prolonged fasting (301).
B. Roles of SIRT3 in Skeletal Muscle
Skeletal muscle plays an important role in the regulation of
lipid metabolism, as lipid catabolism provides a high percent-
age of the energy usage for resting muscle. Similar to its regu-
lation in the liver, SIRT3 levels are decreased in the muscle of
mice fed HFD, whereas they are increased after fasting and
caloric restriction (242). In addition to its regulation by nutri-
tional status, muscle SIRT3 is also modulated by exercise, as
trained mice showed higher SIRT3 levels compared with non-
exercisedmice(139,242) (FIGURE 7).Furthermore,thelackof
Fatty acid oxidation
Metabolic roles of SIRT3 in peripheral tissues and the central nervous system.
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SIRT3 inactivates AMPK and pCREB, leading to the inhibi-
tion of PGC-1? activity in muscle (242).
C. Roles of SIRT3 in Adipose Tissue Function
SIRT3 has been detected at high levels in brown adipose
tissue, while lower levels were found in white adipose tissue
restriction and cold exposure, and increased temperatures
reverse the higher levels of SIRT3 (136, 300) (FIGURE 7).
Furthermore, constitutive expression of SIRT3 in brown
adipocytes increases the expression of PGC-1? and UCP1,
leading to higher thermogenesis and oxygen consumption
VIII. GENETIC MODELS OF SIRT1 AND
SIRT3 UNCOVER NEW ROLES IN
METABOLIC CONTROL AND
A. SIRT1 Gain- and Loss-of-Function Models
and Metabolic Dysfunction
1. Global SIRT1 deficiency affects metabolic control
Early studies of lower organisms lacking Sir2 demonstrated
that Sir2 deficiency eliminates the life-extending effects of
calorie restriction in yeast (200) and decreases life span of
flies (18). In SIRT1-deficient mice, the results are somewhat
controversial, and different phenotypes have been found.
Inbred SIRT1-null mice were born underweight and
showed early postnatal lethality (214). Specifically, SIRT1
knockouts died between E9.5 and E14.5 and exhibit prob-
lems with proper DNA repair and histone modulation
tant developmental defects of the retina and heart (78).
Male (inbred) SIRT1-null mice were shown to be sterile,
potentially due to reduced expression (and thus signaling)
of hypothalamic gonadotropin-releasing hormone and lu-
teinizing hormone (179) and thus attenuated spermatogen-
esis (67). Nevertheless, approximately half the SIRT1-null
mice on an outbred background survived past adulthood
(214). Outbred SIRT1-null mice were visibly smaller (25%)
than their littermates at 2–4 mo, and this difference peaked
(40%) when the mice were 13–20 mo old (45). The surviv-
ing mice have a characteristic defect in eyelid development
rendering them sightless (214). This body weight difference
was not due to food intake, which was similar to that of
wild-type mice; in fact, SIRT1-null mice were hyperphagic
when food intake was normalized to body weight. How-
ever, these mice were hypermetabolic, contained inefficient
liver mitochondria, and had elevated rates of lipid oxida-
When the caloric intake was restricted 40%, SIRT1 knock-
wild-type mice, and their metabolic rate was lower (45).
These findings are similar to those observed after 9 mo of
CR, in which SIRT1 null mice failed to increase their phys-
ical activity, even though they performed better than wild-
type mice on an accelerating treadmill or rotaroad (75).
Importantly, CR did not extend the life span of SIRT1-null
mice (45). Notably, although mice lacking SIRT1 had re-
duced levels of markers of oxidative damage in the brain,
the life span of these mice was shorter under both normal
and caloric restricted conditions (196). In mice, life span is
often determined by the individuals and strain-specific sus-
ceptibility to develop tumors, and SIRT1 might increase life
span by decreasing tumor rates. However, controversy also
surrounds the functions of SIRT1 on tumorigenesis. While
at an increased rate relative to wild-type mice (348), subse-
tumors at normal rates, and resveratrol treatment did not
have a protective effect against tumorigenesis (44).
The complete lack of SIRT1 has differential metabolic ef-
fects on glucose homeostasis and lipid metabolism. Studies
by Bordone et al. (47) showed that SIRT1 deficiency seems
to improve glucose homeostasis. Global SIRT1-knockout
mice demonstrated improved glucose tolerance with de-
creased levels of insulin and blood glucose (47). SIRT1 sup-
pressed UCP2, which inhibited insulin secretion. Accord-
ingly, SIRT1 knockout mice showed high UCP2 expression
(47). It is possible, though, that an improvement in glucose
homeostasis could be due simply to the reduced body
weight, fat mass, and size of the SIRT1-knockout mice. On
the other hand, partial lack of SIRT1 (SIRT1?/?mice) led
to liver steatosis when mice were fed a medium-fat diet
(356). The steatosis was accompanied by an elevated liver-
to-body ratio, liver size, and hepatic lipid deposition (356).
Therefore, it seems that the disruption of SIRT1 activity
may cause beneficial effects on glucose, but it favors lipid
deposition in the liver.
Li et al. (194) determined that global SIRT1 deficiency also
affects cholesterol regulation. The LXRs are important for
cholesterol and triglyceride homeostasis (194). SIRT1-null
mice demonstrated abnormal cholesterol homeostasis due
to the decreased expression of LXR targets and, as a con-
sequence, a reduction of reverse cholesterol transport as
well as high-density lipoproteins (194), but low-density li-
poprotein cholesterol was unchanged compared with wild-
type mice (194).
2. Liver-specific SIRT1 knockdown affects metabolic
The metabolic consequences of SIRT1 deficiency in liver-
specific SIRT1-KO mice are even more variable and dis-
puted than those of global SIRT1-knockout mice. In 2007,
Rodgers and Puigserver (276) observed that hepatic SIRT1
knockdown in mice led to mild hypoglycemia, decreased
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glucose production, increased insulin sensitivity, decreased
blood cholesterol levels, and increased levels of cholesterol
and free fatty acids in the liver during fasting. These results
were consistent with SIRT1 regulation of gluconeogenesis,
cholesterol homeostasis, and free fatty acid oxidation in the
liver (276). Most of these results were observed only under
fasting conditions (276), and the overexpression of SIRT1
reversed many of these changes. However, another study
suggests that SIRT1 knockdown in the liver may be benefi-
cial in a rat model of type 2 diabetes (96). The knockdown
of SIRT1 in those rats improved insulin sensitivity, lowered
fasting glucose levels, decreased hepatic glucose produc-
tion, and decreased plasma total cholesterol levels (96).
Studies in liver-specific SIRT1-knockout mice on a high-
some protection from accumulating fat, compared with
wild-type mice. In contrast, while under calorie restriction,
the liver-specific KO mice displayed the same phenotype as
wild-type mice. These observations suggested that in nor-
mal mice hepatic SIRT1 may be inactivated as a result of a
calorie-restricted diet and activated as a result of a high-
calorie diet (74). As discussed before, these results are in
direct contrast to the dogma that calorie restriction univer-
sally increases SIRT1 activity, and new questions are
brought to light concerning the unique role of hepatic
SIRT1 (74). Contradicting the above findings, a 2009 study
by Purushotham et al. (259) suggested that hepatic SIRT1
knockout was harmful for mice that were challenged with a
high-fat diet. These mice developed many metabolic prob-
lems including liver steatosis, hepatic inflammation, and
endoplasmic reticulum stress (259). Accordingly, later re-
search indicated that the specific deletion of hepatic SIRT1
in mice impaired the mTorc2/Akt signaling and resulted in
hyperglycemia, oxidative damage, and insulin resistance
(347). In this sense, the hepatic overexpression of SIRT1 in
mice ameliorated insulin resistance and restored glucose
Overall, based on the existing evidence collected in SIRT1-
SIRT1 in regulating glucose, cholesterol, and lipid metabo-
lism. Species differences, methodological differences (viral
knockdown vs. germline mutation), the overall problem of
genetic background (inbred vs. outbred), and the compro-
mised health of SIRT1-null mice are factors that make it
hard to evaluate the role of hepatic SIRT1.
3. Beneficial effects of global SIRT1 overexpression
on systemic metabolism
As happened with global SIRT1 deletion, the overexpres-
sion of SIRT1 in mice also showed different results with
respect to body weight and feeding behavior. However,
most studies show compelling evidence that SIRT1 overex-
pression offers substantial benefits on serum cholesterol
and insulin levels and increased resistance to high-fat diet
induced glucose intolerance and insulin resistance (28, 46,
249). Mice overexpressing SIRT1 under the control of a
than their standard-diet fed littermates. In addition, they
had lower cholesterol, insulin, and fasting glucose levels as
transgenic mice performed better on a rotarod assay (46).
All these characteristics resembled the phenotype showed
by mice under CR. However, subsequent studies in mice
that overexpressed SIRT1 under its own promotor showed
slightly different results (28, 249). Banks et al. (28) showed
that on a standard diet SIRT1 transgenic mice had normal
insulin sensitivity but decreased food intake and locomotor
activity, resulting in decreased energy expenditure but nor-
mal body weight. Concomitant overexpression of SIRT1 in
environmental and genetic models of insulin resistance and
diabetes improved glucose tolerance due to decreased he-
patic glucose production and increased adiponectin levels;
however, body weight and body composition remained un-
changed (28). Accordingly, SIRT1 overexpression did not
protect mice from high-fat diet-induced obesity (28, 249).
However, contrary to previous studies, SIRT1 transgenic
mice displayed higher energy expenditure, which was com-
pensated for by a parallel increase in food intake (249).
Nevertheless, in agreement with previous studies, high-fat
diet fed SIRT1 overexpressing mice displayed lower lipid-
induced inflammation and better glucose tolerance and
were protected from hepatic steatosis (249). The mecha-
nisms modulating these actions involved the induction of
antioxidant proteins MnSOD and Nrf1, possibly via stim-
ulation of PGC-1?, and a lower activation of proinflamma-
tory cytokines, such as tumor necrosis factor-? and inter-
leukin-6, via downmodulation of NF-?B activity (249). The
overexpression of SIRT1 in mice also potentiated the stim-
ulation of fatty acid oxidation and energy expenditure
through its NAD?-independent actions on the cAMP/PKA
4. Pancreatic ?-cell-specific SIRT1 overexpression
improves glucose homeostasis
In RINm5F cells or isolated rat islets, the pharmacological
stimulation and ectopic expression of SIRT1 completely
prevented cytokine-mediated cytotoxicity, indicating that
SIRT1 participates in the maintenance of normal insulin-
secreting responses to glucose in isolated rat islets (192). In
addition, SIRT1 positively regulated insulin secretion in
pancreatic ?-cells by repressing UCP2 (47). These results
were later corroborated in mice with genetic manipulation
of SIRT1 in the pancreas. For instance, the Imai laboratory
has studied beta cell-specific SIRT1-overexpressing (BE-
STO) transgenic mice over the past few years (225, 269). In
these mice, the higher dosage of SIRT1 potentiated glucose-
(225), potentially via SIRT1-mediated inhibition of UCP2
(225). However, when BESTO mice were ageing to 18–24
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mo, this beneficial glucose-stimulated insulin secretion was
B. SIRT3 Gain- and Loss-of-Function Models
and Mitochondrial Function
Mice deficient in SIRT3 were viable and did not show any
metabolic disorder. They had normal body weight, body
composition, oxygen consumption, respiratory quotient,
and physical activity (207). They also responded normally
to food deprivation, and adaptive thermogenesis was not
altered (207). However, the lack of SIRT3 induced global
mitochondrial lysine acetylation through the maintenance
of basal ATP levels (5, 171, 207). Another important effect
of the global deficiency of SIRT3 was that these mice had
increased stress-induced superoxide levels and genomic in-
stability, leading to a tumor-permissive phenotype (171).
Prolonged SIRT3 expression in the mitochondria of murine
adipocytes potentiated cellular respiration, lowered mem-
brane potential, and attenuated reactive oxygen species
In addition to modulating metabolism, SIRT3 controls mito-
the regulatory component of the mPTP cyclophilin D (122).
dependent increase in mPTP opening and accelerated signs of
aging in the heart, known phenotypes of excessive mPTP
opening. Knockout mice experience cardiac hypertrophy and
fibrosis at 13 mo of age and are also sensitive to transverse
aortic constriction, as evidenced by cardiac hypertrophy, fi-
mouse KO show that SIRT3 is critical for delaying mitochon-
drial dysfunction during aging.
C. SIRT1 and the Molecular Clock
that closely resembles the 24-h daily cycle, even in constant
light conditions (320). This autonomous circadian oscilla-
tion is closely connected to the body’s metabolism, and
alterations in the circadian cycle can have an enormous
impact on metabolism. For instance, earlier reports had
shown that dysregulation of circadian rhythms leads to
other metabolic problems (331). Notably, recent reports
also showed that global absence of SIRT1 led to dysregula-
tion of circadian rhythms (352). Thus, in the following
rhythms, NAD?, and metabolic dysfunction are discussed
in more detail.
1. Molecular underpinnings for a circadian clock
In 1994, Takahashi and colleagues (62) identified the pro-
tein CLOCK as main regulator of the autonomous circa-
dian rhythm in cells. CLOCK is a transcription factor from
the group of bHLH (basic helix-loop-helix) proteins that
dimerizes with BMAL1 to regulate the transcription of sev-
eral circadian genes (e.g., Cryptochrome and Period genes)
(17, 26, 51). Accumulation of the transcripts (Cry and Per)
facilitates complex formation with CLOCK-BMAL1, si-
lencing the transcription of circadian genes (31, 61, 69).
Interestingly, CLOCK is a histone acetyltransferase (13)
that directly acetylates histone H3 and its partner BMAL1.
Moreover, when Per2 binds to the CLOCK-BMAL1 com-
plex, it is also acetylated (2). These acetylation reactions are
essential for the binding and regulation of the transcription
of the circadian genes (39).
2. Circadian fluctuations in NAD?levels fine-tune
the circadian oscillation of the CLOCK-BMAL1
complex via Sirt1
Recently, studies in mammalian cells suggest that NAD?
levels are regulated in a circadian rhythm. This rhythm is
due to the salvage pathway, a two-step reaction that
converts nicotinamidetonicotinamide mononucleotide
(NMN) to NAD?(210, 228, 270). The two enzymes
responsible for catalyzing these reactions are nicotin-
amidephophoribosyltransferase (NAMPT) and NMN
adenylytransferases (NMNAT) (95, 273). What makes
this scenario interesting for the understanding of sirtuins’
physiology is that SIRT1 utilizes NAD?in its biochemi-
cal reaction to generate nicotinamide. Thus NAMPT,
NMNAT, and SIRT1 close a loop in a biochemical cas-
cade from nicotinamide down to NMN, NAD?, and fi-
nally nicotinamide (228, 270).
ing to the circadian cycle, then SIRT1 activity should vary
as well, because it is linked to NAD?levels. Several reports
shed light on these mechanisms, showing that SIRT1 inter-
acts with CLOCK to generate a protein complex (SIRT1-
CLOCK). SIRT1 was shown to bind and deacetylate the
CLOCK complex, leading to degradation of the complex
and inactivation of circadian gene transcription (2, 39).
Thus SIRT1 deacetylase activity affects the CLOCK com-
plex by regulating acetyl residues, driving degradation of
the complex. Additionally, the CLOCK-BMAL1 com-
plex was shown to bind to the NAMPT promoter where
it upregulates NAMPT expression (40, 47). Increases in
NAMPT levels contribute to enhance the levels of NAD?,
which in turn activates SIRT1. SIRT1 subsequently
deacetylates the CLOCK complex and releases it from the
DNA, decreasing the transcription of NAMPT. With less
NAMPT, levels of NAD?decrease and, consequently, so
does SIRT1 activity. This NAMPT-SIRT1 loop acts to
fine-tune the circadian oscillation of the CLOCK-
BMAL1 complex, linking metabolism variances to the
circadian rhythm (40, 47).
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3. Circadian rhythm control by SIRT1 may be
mediated by the central nervous system
The master clock of mammals is located in the suprachias-
matic nucleus (SCN) of the basal hypothalamus (349). Le-
sion of the SCN abolished the cyclic nature of locomotor
activity in rodents (147–149, 223, 312, 313). Transplanta-
tion of SCN grafts to the lesioned SCN could restore the
oscillatory behavior in lesioned animals (264, 265, 305).
The nature of the oscillatory behavior of the transplanted
graft was located intracellularly, as evidenced by the fact
that when neurons from the SCN are dissociated in cell
culture their oscillatory (circadian) activity is maintained
for many days. Thus it is well accepted that the SCN repre-
sents the dominant molecular clock that orchestrates the
body’s circadian rhythm based on the Zeitgeber light.
SIRT1 is expressed in the SCN, next to the ubiquitously ex-
pressed CLOCK, BMAL1, and all other components of the
has not been identified in brain tissue (48). Nevertheless, spe-
cific groups of neurons could express minute amounts of
NAMPT, which could be sufficient to regulate SCN clock
open question of NAMPT expression in brain or the SCN,
warrants further research. Ultimately, despite the dominant
metabolic oscillations, NAD?, and SIRT1 in the CNS and
specifically in the SCN remain unclear.
4. SIRT1 and the molecular clock in peripheral tissues
Even though the SCN maintains the body’s circadian rhythm,
several other tissues maintain an independent, cell-autono-
mous oscillatory rhythm, which in most cases resembles the
the light is the main circadian regulatory input, in peripheral
tissues cellular energy metabolism (feeding) serves as a potent
synchronizer of subsidiary oscillations. Since cellular energy
status directly affects NAD levels and thus SIRT1 activity,
cell-autonomous molecular interactions of SIRT1 with
CLOCK in peripheral organs may play a major role in circa-
dian gene oscillation. Indeed, although most findings on the
role of SIRT1-CLOCK were derived from studies in mamma-
lian cells, most have also been confirmed in peripheral tissues
such as liver (17, 227). In conclusion, there is compelling evi-
dence that SIRT1 acts as a metabolic rheostat for oscillatory
rhythms in peripheral organs.
D. SIRT1 and Diabetes-Induced Cardiac
SIRT1 conferred cardioprotection in the heart (8, 9, 252)
during oxidative stress (251). Resveratrol, a proposed
SIRT1 activator (reviewed in section X), prevented hypox-
ia-induced apoptosis in cardiomyocytes through SIRT1-
mediated FOXO1 regulation (73). SIRT1-mediated cardiac
moderate; in contrast, high doses of SIRT1 exacerbated
oxidative stress and induced cardiomyopathy (8).
Hyperglycemia during diabetes can cause significant car-
diac deterioration and dysfunction. Recent studies have
demonstrated that diabetes-induced cardiac dysfunction
may be alleviated through activation of the SIRT1 pathway
(89, 316, 333). SIRT1 regulated angiogenic activity in en-
dothelial cells by deacetylating and inhibiting FOXO1
(257), which is a negative regulator of angiogenesis (112,
258). In the diabetic state, repression of FOXO1 activity
causes endothelial dysfunction and diminished angiogene-
sis. A study by Balestrieri et al. (26) proposed that SIRT1 is
an important regulator of endothelial progenitor cell dys-
function in a high-glucose environment. When endothelial
progenitor cells were exposed to a high-glucose environ-
ment, downregulation of EPC activity correlated with re-
duced SIRT1 expression and elevated acetyl-FOXO1 ex-
pression (26). In addition to these effects on endothelial
progenitor cells, SIRT1 has been implicated in cardiac dys-
function. Cardiac dysfunction in diabetic cardiomyopathy
reduced sarcoplasmic calcium ATPase (SERCA2a) expres-
sion levels. Sulaiman et al. (316) showed that activation of
SIRT1 by resveratrol increased levels of SERCA2a mRNA
and improved cardiac function in mice injected with strep-
tozotocin, a chemical used to model type 1 diabetes in ani-
According to Orimo et al. (237), the mechanism of SIRT1’s
beneficial action was through p53 regulation. The authors
describe that hyperglycemia induced vascular senescence
unless SIRT1 was introduced or p53 was removed; activa-
tion of SIRT1 by resveratrol lessened the extent of vascular
dysfunction in diabetic mice, specifically through the
SIRT1-p53 pathway (237).
IX. GENETIC POLYMORPHISMS OF SIRT1
AND SIRT3 IN HUMANS
A. Genetic Polymorphisms of SIRT1
Very little is known about the genetic variation of SIRT1
and its effects on energy homeostasis in humans, and the
the relationship between variants in SIRT1 and obesity in
1,068 obese patients and 313 normal-weight control sub-
jects (248). The authors found that males but not females
with a SIRT1 single nucleotide polymorphism (SNP) were
associated with increased visceral adiposity (248). A similar
study using a large sample-size population of 6,251 elderly
subjects (population-based Rotterdam Study) found that
there were two variants in SIRT1 that were associated with
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weight gain (385). In a smaller study using 389 patients
with metabolic syndrome and 547 controls, a significant
association between one SIRT1 SNP and metabolic syn-
drome was found (68). However, another study performed
in 917 overweight individuals investigating the associations
of SIRT1 SNPs with metabolic response did not find an
association of SIRT1 SNPs with baseline BMI or with BMI
change after a 9 mo follow-up (351).
The genetic variation of SIRT1 and its effects on human
longevity are also poorly studied. One report using DNA
from 1,573 long-lived individuals (centenarians and nona-
genarians) found no evidence between the five SNPs ana-
lyzed and longevity (103). Similar findings were observed in
another population-based Leiden85-plus Study of 1,245
subjects (183). Therefore, it seems that SNPs for SIRT1 are
not related with human longevity, but further studies using
larger-size populations are needed to elucidate this issue.
B. Genetic Polymorphisms of SIRT3
The knowledge about the potential role and genetic altera-
tions of SIRT3 in humans is even lower than for SIRT1.
SIRT3 is located at the telomeric terminal on 11p15.5 chro-
mosome, and studies performed in people over 100 years
have demonstrated that there is a correlation between lon-
gevity and polymorphism of four genes located in this re-
gion (85). The researchers found a SNP marker in exon 3 of
the SIRT3 gene, and there was a male-specific relationship
between this marker and longevity (278). The same labora-
tory discovered a VNTR polymorphism in intron 5 of the
SIRT3 gene (36). Moreover, by analyzing allele frequencies
as a function of age, the researchers found that the allele
years (36). Thus this study suggests that low expression of
SIRT3 is detrimental for longevity in humans. Another
study using 640 individuals found two human SIRT3 SNPs
and suggested that SIRT3 increased cellular respiration
and age and between SIRT3 and endurance exercise (190).
The results showed that the protein levels of SIRT3 in mus-
cle were lower in old sedentary subjects compared with
young sedentary subjects (190). Although not statistically
significant, this study suggests that endurance-trained indi-
viduals might be protected against diminishing levels of
X. METABOLIC CONSEQUENCES OF
PHARMACOLOGICAL SIRT1 ACTIVATION
A. Resveratrol: A Specific SIRT1 Activator?
Early studies showing the role of Sir2/SIRT1 in the mecha-
nisms responsible for the effects of calorie restriction in
prolonging life span (165, 200, 327) raised the possibility
that SIRT1 could be implicated in glucose metabolism and
insulin sensitivity, key factors impaired during aging. Initial
screening studies identified the polyphenol resveratrol as a
to stimulate SIRT1 activity in vivo, several groups reported
benefits of resveratrol in models of metabolic disorders (34,
186). Resveratrol administration was shown to protect
glucose intolerance, insulin resistance, or life span reduc-
tion, potentially through activation of SIRT1, although no
beneficial effects on body weight could be observed (6, 34,
186). However, other studies failed to show activation of
SIRT1 by resveratrol (163, 239) and suggested that previ-
ous reports on the activation of SIRT1 by resveratrol could
be due to an interaction between resveratrol and the fluo-
rophore used in the assay (163, 239). This suggestion was
challenged by a subsequent report showing that the fluoro-
phore is dispensible for SIRT1 activation to occur (80).
Instead, the authors suggest that the fluorophore mimics a
bulky-hydrophobic amino acid in natural substrates (80).
Resveratrol has been shown to be an indirect activator of
several proteins such as AMPK, an important enzyme for
energy and glucose homeostasis (34, 70, 84, 146, 244, 296,
332). AMPK senses the levels of ATP in the cell milieu.
Metformin, a well-known activator of AMPK, is widely
used to treat type 2 diabetes. Several reports showed that
AMPK and SIRT1 share similar molecular pathways, and
activation of SIRT1 by resveratrol could be a consequence
of AMPK activation (332). Indeed, it was recently reported
that the effects of resveratrol improving metabolic param-
eters in models of metabolic disorders are dependent on the
expression of the AMPK subunit ? (332). In this report, the
authors tested the effects of resveratrol in AMPK?1 and
AMPK?2 KO mice; resveratrol failed to improve insulin sen-
sitivy, glucose tolerance, and mitochondria biogenesis in an
AMPK-dependent manner, and failed to decrease fat mass.
The authors also showed that resveratrol increased the
ity (332). This effect was also dependent on the expression of
AMPK, thus shedding light on a possible indirect mechanism
by which SIRT1 is activated by resveratrol through modula-
tion of NAD?/NADH levels via AMPK (57).
On the other hand, SIRT1 regulates LKB1, an upstream
regulator of AMPK (140, 386). Thus the effects of resvera-
down of LKB1 reduces the ability of resveratrol to protect
cells from mitochondrial dysfunction (302). Clearly, the
interplay between SIRT1 and AMPK is complex, and it is
not possible to conclude whether resveratrol acts on AMPK
independently of SIRT1. The testing of resveratrol in a
SIRT1 KO mouse would likely clarify this debate (for fur-
ther review, see Refs. 56, 123).
Interestingly, considerable insight can be derived from a
randomized double-blind crossover study in healthy, obese
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men treated with resveratrol (326). In this study resveratrol
significantly reduced sleeping and resting metabolic rate.
intrahepatic lipid content, circulating glucose, triglycerides,
alanine-aminotransferase, and inflammation markers; im-
proved systolic blood pressure; and decreased the HOMA
index (326). Therefore, this study suggests that the treat-
ment with resveratrol induces metabolic changes in obese
humans mimicking the effects of calorie restriction.
B. Natural SIRT1 Activators
It is well known that nicotinamide adenine dinucleotides
(NAD?and NADH) are essential mediators of energy ho-
meostasis (39). Consequently, increased intracellular levels
of NAD?activate sirtuin-dependent metabolic control.
Thus compounds that modulate NAD?/NADH ratio are
likely to also exert effects on SIRT1-medulated metabolic
control. Indeed, ?-lapachone, an o-naphthoquinone ex-
tracted from bark of the lapacho tree (Tabebuiaavellane-
dae), has been shown to stimulate NADH oxidation
through interaction with NADH:quinoneoxidoreductase 1
(NQO1); treatment of diet-induced obese and leptin-defi-
marized as metabolic syndrome, such as increased adipos-
ity, glucose intolerance, dyslipidemia, and fatty liver (145).
Therefore, the pharmacological administration of ?-lapa-
chone mimics effects of SIRT1 activation/overexpression,
and as a matter of fact, the treatment with ?-lapachone
increased SIRT1 mRNA expression in muscle and white
adipose tissue (145).
Kaempferol, a flavonoid with anti- and pro-oxidant activity
present in various natural sources, was shown to activate
SIRT3 in myelogenous leukemia cell line K562 and promy-
elocitic human leukemia U937 (213). Consistently, down-
regulation of SIRT3 in these cell lines abolished the actions
of Kaempferol (213). Since Kaempferol induced apoptosis,
it seems possible that SIRT3 might play an important func-
tion in the mitochondrially mediated apoptosis.
C. Novel Synthetic SIRT1 Activators
Improve Metabolic Control
After the discovery of resveratrol as a SIRT1 activator, sev-
eral synthetic small molecule activators of SIRT1 were re-
ported (220). These molecules, 1,000 times more potent
than resveratrol but structurally distinct (220), were shown
to act through the same enzymatic mechanism, i.e., binding
to an allosteric site exposed in the enzyme-substrate com-
exhibited good oral bioavailability in rodents, and their
administration substantially improved both glucose and in-
sulin homeostasis in ob/ob mice, diet-induced obese mice,
and Zucker fa/fa rats (220). Consistent with these findings,
the same researchers have shown that these activators reca-
pitulate many of the molecular events downstream of CR in
vivo, such as enhancing mitochondrial biogenesis, improv-
ing metabolic signaling pathways, and blunting proinflam-
matory pathways in mice fed a high-fat, high-calorie diet
(308). Similarly, an independent group treated mice with
nonalcoholic fatty liver disease (NAFLD) with one of these
activators (SRT1720) and found that the compound re-
duced the expression of lipogenic enzymes, serum lipid pro-
files, expressions of marker genes for oxidative stress, and
inflammatory cytokines in the liver of these mice (360).
SRT1720 administration was further shown to enhance en-
durance and protect from diet-induced obesity and insulin
resistance, potentially by enhancing oxidative metabolism
in skeletal muscle, liver, and brown adipose tissue (99). In
vitro studies using SRT1720 have shown that this drug
increases insulin-stimulated glucose uptake in adipocytes
However, a recent study attributed activation of SIRT1 to
drug binding to the fluorphore, casting doubts on the spec-
ificity of these molecules (239). In contrast to the two inde-
pendent studies, in vivo administration of SRT1720 was
lethal to mice and failed to decrease plasma glucose or im-
prove mitochondrial capacity in mice fed a high-fat diet,
although insulin levels were decreased (239). This report
concludes that these synthetic activators, as well as resvera-
trol, exhibit multiple off-target activities against receptors,
enzymes, transporters, and ion channels and, therefore, are
not direct activators of SIRT1 (239). Since this report, ac-
tivation of SIRT1 has been shown to occur on natural pep-
tides, and the enzyme kinetics for activation by SRT1720
and other small molecules are consistent with an allosteric
activation mechanism (80).
XI. FUTURE PERSPECTIVES
Decades of biomedical progress and vast improvements
in our living conditions allow us to lead productive lives
into old age. At the same time, we have to face unprece-
dented negative impacts of our Western life-style, result-
ing in the rapid rise in obesity and its comorbidities,
diabetes and fatty liver disease. Although major efforts
are being made to attenuate such negative impacts of our
Western life-style on obesity and its sequelae, to date
there are no efficient pharmacological treatment options
for weight control available, and surgical options such as
bariatric surgeries, albeit being highly efficient, may not
(yet) be feasible for large proportions of the affected
population. In addition, although changes in life-style
may hold the greatest promise to avoid obesity and met-
abolic disorders for a majority of us, dietary regimens
such as calorie restriction and exercise have repeatedly
been shown not to lead to adequate weight loss if not
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continued with highest perseverance and diligence. Fur-
thermore, drugs that can mimic caloric restriction hold
the promise of being able to prevent and treat numerous
other diseases such as cancer and heart disease.
Although pharmacological or genetic activation of sirtuins,
and particularly SIRT1, resembles the beneficial effects of
caloric restriction, making them attractive drug targets, we
should not forget that sirtuins act on many different tran-
scription factors, which are involved in numerous biologi-
cal activities. Clinical trials will be necessary to address if
SIRT1 analogs have beneficial effects in obese and/or dia-
SIRT3 is still a largely unexplored drug target. Although
there are promising in vitro studies overexpressing SIRT3,
to our knowledge, there are no specific SIRT3 activators
available, and its potential pharmacological actions in vivo
remain completely unknown.
It is certain that new targets modulated by SIRT1 and
SIRT3 will appear in the near future, and some of those
targets will be important for several metabolic actions. To
precisely dissect the molecular pathways modulating sir-
tuins is a crucial step in elucidating the real potential of
these deacetylases, and will require years of investigation.
With the information obtained on sirtuins to date, it seems
that this big challenge deserves to be weighed out, and we
will certainly enjoy more exciting studies on this topic dur-
ing the next years.
Address for reprint requests and other correspondence:
M. H. Tschöp, Institute for Diabetes and Obesity, German
Center for Diabetes Research, Helmholtz Center Munich,
German Research Center for Environmental Health
(GmbH), Ingolstaedter Landstr. 1, 85764 Neuherberg/Munich,
Germany (e-mail: firstname.lastname@example.org).
No conflicts of interest, financial or otherwise, are declared
by the authors.
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