ArticlePDF AvailableLiterature Review

Sirtuins: The ‘ magnificent seven ’, function, metabolism and longevity

  • Centre de Recherche des Cordeliers (CRC), Paris, France


The sirtuin family of histone deacetylases (HDACs) was named after their homology to the Saccharomyces cerevisiae gene silent information regulator 2 (Sir2). In the yeast, Sir2 has been shown to mediate the effects of calorie restriction on the extension of life span and high levels of Sir2 activity promote longevity. Like their yeast homologs, the mammalian sirtuins (SIRT1-7) are class III HDACs and require NAD(+) as a cofactor to deacetylate substrates ranging from histones to transcriptional regulators. Through this activity, sirtuins are shown to regulate important biological processes ranging from apoptosis, adipocyte and muscle differentiation, and energy expenditure to gluconeogenesis. We review here the current knowledge regarding the role of sirtuins in metabolism, longevity, and discuss the possible therapeutic applications that could result from the understanding of their function in different organs and pathologies.
Sirtuins: The magnificent seven’, function, metabolism and longevity
Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire de Strasbourg (IGBMC), INSERM/CNRS/ULP, Illkirch,
Institut Clinique de la Souris, Genopole, Illkirch, France,
Laboratoire de Biochimie Ge´ne´rale et Spe´cialise´e,
Hoˆpitaux Universitaires de Strasbourg, Strasbourg, France, and
Service de Neurochirurgie, Hoˆpital de Hautepierre, C.H.U.
de Strasbourg, Strasbourg, France
The sirtuin family of histone deacetylases (HDACs) was named after their homology to the Saccharomyces cerevisiae gene
silent information regulator 2 (Sir2). In the yeast, Sir2 has been shown to mediate the effects of calorie restriction on the
extension of life span and high levels of Sir2 activity promote longevity. Like their yeast homologs, the mammalian sirtuins
(SIRT1-7) are class III HDACs and require NAD
as a cofactor to deacetylate substrates ranging from histones to
transcriptional regulators. Through this activity, sirtuins are shown to regulate important biological processes ranging from
apoptosis, adipocyte and muscle differentiation, and energy expenditure to gluconeogenesis. We review here the current
knowledge regarding the role of sirtuins in metabolism, longevity, and discuss the possible therapeutic applications that
could result from the understanding of their function in different organs and pathologies.
Key words: Calorie restriction, longevity, metabolism, SIRT
Histone acetylation is the main type of covalent
histone modification and is achieved by a class of
enzymes termed histone acetyltransferases (HATs)
(1). HATs acetylate histones on lysines, whereas
histone deacetylation involves another family of
enzymes, the histone deacetylases (HDACs) ((2), a
review). These HDACs are classified in three groups
on the basis of their homology with the yeast
Saccharomyces cerevisiae HDACs: RPD3 (group I),
HDA1 (group II), and Sir2 (group III). Class I and
II enzymes are inhibited by trichostatin (TSA),
whereas class III HDACs are not inhibited by TSA
and are NAD
-dependent (3,4), suggesting a reg-
ulation of Sir2 molecules by the metabolic state of
the cells (reviewed in (5)). Since the Sir2 family of
proteins are also able to exert their enzymatic activity
not only on histones but also on numerous other
proteins, such as the transcriptional regulators, they
are involved in many cellular processes, ranging in
yeast from gene silencing, DNA repair, progression
of the cell cycle, to the control of ageing.
The Sir2 protein first deacetylates histones and
then localizes with the yeast protein Sir4, to form a
tight silencing complex binding to the telomeres (6).
The localization of Sir2 complex to the histone tails,
initially identified to induce transcriptional repres-
sion of the silent mating type loci HML and HMR
(homothallic mating-type loci left and right, respec-
tively) (7,8), produces chromatin silencing, and a
recent study reported that the Sir2 complex reduces
the promoter occupancy by the transcription factors
IIB and IIE (TFIIB, TFIIE) and the RNA poly-
merase II (Pol II) preventing hence the proper
assembly of the preinitiation complex of the tran-
scriptional machinery (9). Most interesting was the
fact that overexpression of the gene encoding the
Sir2 protein leads to a decrease in histone acetylation
(10) and an increase in life span in yeast (11), in the
nematode Caenorhabditis elegans (12) and in metazo-
ans (13). Likewise, Sir2-activating compounds
Correspondence: Johan Auwerx, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire de Strasbourg (IGBMC), INSERM/CNRS/ULP, 1 rue
Laurent Fries, BP 10142, 67404 Illkirch, France. E-mail:
Annals of Medicine. 2007; 39: 335–345
ISSN 0785-3890 print/ISSN 1365-2060 online # 2007 Taylor & Francis
DOI: 10.1080/07853890701408194
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(STACs), such as resveratrol, present in the skin of
red grapes, promote longevity in yeast (14) and other
organisms ranging from the worm and drosophila
(15) to the mouse (16). On the other hand, both
mutations of the Sir2 gene and pharmacological
inhibition of Sir2 protein by nicotinamide induces an
acceleration of ageing in yeast (17). Although the
functions of Sir2 have been relatively well estab-
lished in yeast and C. elegans, their function in
mammals remains rather elusive. This review will
give an overview of the role of the sirtuin family of
proteins in different species and their potential
contribution to disease.
SIRT1/Sir2, caloric restriction, metabolism,
neurodegeneration, and longevity
In the last few years, an increasing number of studies
from S. cerevisiae, C. elegans, Drosophila melanogaster,
and mouse models have linked caloric restriction
(CR) and metabolism with longevity. The matter
has been extensively reviewed by Bordone and
Guarente (18); in this section we give an overview
of how SIRT1, the mammalian Sir2 homolog, which
is the best characterized sirtuin family member, and
CR could affect metabolism and longevity in
humans. To better understand the mechanisms by
which CR extends life span, it is instructive to take a
close look at studies carried out in various species.
Studies based on non-mammalian models
Studies in Saccharomyces cerevisiae
In yeast, reduction in glucose levels in the media,
mimicking CR, results in a substantial extension in
life span, which is Sir2p- and NAD
since mutants for Sir2p and nicotinate phosphor-
ibosyl transferase (NPT1), an enzyme required for
formation, failed to reproduce this effect
(19). In the same study, mutants for components of
the glucose-signaling pathway, such as the GTP-
GDP (guanosine triphosphate-guanosine dipho-
sphate) exchange factor CDC25, the glucose sensing
receptors gpr1 and gpa2, and hexokinase, the first
enzyme in the glycolytic pathway, mimicked the CR-
mediated increase in longevity. This life span
extension due to CR was attributed to an increase
in respiration (20). Indeed a mutation in the gene
encoding the cytochrome c1 CYT1 is no longer able
to promote CR-mediated longevity, suggesting that
blocking the mitochondrial electron transport chain
and respiration prevents life span extension.
Moreover, overexpression of Hap4 in yeast, a gene
known to cause a switch from fermentation to
respiration, yielded a 35% extension in life span
under normal glucose conditions. It is well estab-
lished that respiration increases the NAD
ratio through oxidation of NADH by the NADH
dehydrogenase (21).
The Sir2p-mediated conversion of NAD
leads to
the formation of nicotinamide (NAM), a powerful
Sir2/SIRT1 inhibitor (17) and O-acetyl-ADP-ribose
(O-AA-ribose) (22) (Figure 2A). Recent evidence
demonstrated an active role of O-AA-ribose in the
regulation of gene silencing by the Sir2/3/4 assembly
complex (23). In yeast, an alternative route to
synthesize NAD
, other than its de novo production
from the amino acid tryptophan, and which starts
from nicotinic acid (NA), is present under the form
of the NAD
salvage pathway. In the NAD
pathway, NAM obtained from NAD
cleavage is
deaminated to NA by the nicotinamidase PNC1.
NA formed through the actions of PNC1 then
undergoes a series of reactions giving successively
rise to nicotinate mononucleotide (NAMN), nicoti-
nate adenine dinucleotide (NAAD), and NAD
, that
are catalyzed by the enzymes nicotinate phosphor-
ibosyl transferase (NPT1), nicotinate mononucleo-
tide adenyltransferases (NMAT1/2), and NAD
SIR2 silent information regulator 2
HAT histone acetyltransferase
HDAC histone deacetylase
CR calorie restriction
PKB/AKT protein kinase B
GDP guanosine diphosphate
GTP guanosine triphosphate
nicotinamide adenine
NADH reduced form of NAD
Pol II RNA polymerase II
TFIIB transcription factor IIB
TFIIE transcription factor IIE
Daf-16 ‘dauer’ larvae transcription
FOXO forkhead box subgroup ‘O’
transcription factor
HML homothallic mating-type loci left
HMR homothallic mating-type loci
NFkB nuclear factor kappa B
transcription factor
PGC-1a peroxisome proliferator-activated
receptor gamma (PPARc)
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synthetase (QNS1), respectively (24). The newly
recycled NAD
then serves again as a cofactor for
Sir2p-mediated deacetylation (Figure 2A).
Multiple protagonists of the NAD
salvage path-
way participate in life span extension in yeast.
Furthermore, an intense debate about whether
Sir2p-dependent CR life span extension could be
due to a depletion in the noncompetitive Sir2p
inhibitor NAM, rather than a modification in the
/NADH ratio, is still ongoing. Interestingly,
CR increased the expression of PNC1 and hence
PNC1 activity, which is required and sufficient to
extend life span in yeast through Sir2p activation.
Conversely, mutations in the yeast PNC1 gene
accelerate cellular ageing (25). These observations
hence suggested that NAM depletion, through the
activation of PNC1, is sufficient to activate Sir2 and
increase longevity in yeast. Moreover, mutation of
PNC1 and overexpression of Nnt1, a NAM methyl-
transferase which reduced the excess NAM levels,
Figure 1. Sir2/SIRT1 and signaling pathways in different species. In yeast Sir2p activity is increased through increased expression of the
salvage pathway enzyme, nicotinamidase PNC1, but also by increasing the NAD
/NADH ratio (or lowering NADH levels) in
response to calorie restriction (CR). Mutation of the glucose sensing receptors Gpr1 and Gpa2 mimics the CR effects on Sir2p, which
inhibit the formation of the deleterious extrachromosomal circular DNA repeats (ERCs) and contribute to life span extension in yeast. The
glucose and nutrients fermentation pathway activates the AKT-related kinase Sch9 which induces oxidative stress and participates in yeast
ageing. Inactivation of this pathway promotes longevity in yeast. The longevity pathway is conserved amongst worms, flies and mammals.
Activation of the insulin growth factor receptor IGFR (Daf-2/dIGFR) activates the insulin receptor substrate IRS (p65/CHICO), which
stimulates phosphoinositol-3 kinase PI3K (AGE-1/dPI3K), which in turn phosphorylates the PKB/AKT kinase. AKT phosphorylates and
inactivates FOXO (Daf-16/dFOXO). Inactivation of the insulin receptor pathway promotes longevity in worms, drosophila, and mammals
through increased activity of FOXO. Sir2/SIRT1 activation induces life span extension through interaction with Daf-16/FOXO factors.
Gpr-15G-protein coupled receptor-1; Gpa-25G-alpha protein-2; FGM5Fermentable growth medium; PNC15pyrazinamidase/
Nicotinamidase 1; Sir2p5yeast silent information regulator 2; NADH5reduced form of nicotinamide adenine dinucleotide;
Sch95Saccharomyces cerevisiae protein kinase 9; ERCs5extrachromosomal circular DNA repeats; Daf-165‘dauer’ larvae transcription
factor-16; AGE-15worm homolog of the phosphoinositol-3 kinase; AKT5protein kinase B; Rpd35reduced potassium dependence 3 (class
II histone deacetylase); IGF-15insulin growth factor-1; FOXO5forkhead box subgroup ‘O’ transcription factor; IRS5insulin receptor
substrate; CHICO5 drosophila homolog of IRS; SIRT15sirtuin 1.
Sirtuins and metabolism 337
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restored the CR-induced life span extension in
PNC1 mutants, suggesting that other factors (such
as NADH lowering) might play a role in CR-
mediated life span extension (26). Other compo-
nents of the NAD
salvage pathway, like NPT1, the
rate-limiting enzyme in the NAD
biosynthesis, have
also been shown to regulate life span in yeast.
Increased dosage of NPT1, but not of NMAT,
enhanced the total cellular NAD
levels and
enhanced the transcriptional activity of the catalytic
domain of Sir2p thereby extending yeast life span
(27), whereas NPT1 mutations yielded a defect in
silencing at silent mating-type loci, telomeres, and
rDNA (28). Overexpression of two related mito-
chondrial NADH dehydrogenases, Nde1 and Nde2,
decreased NADH levels without changing NAD
levels, but still increased yeast life span both under
normal glucose culture conditions (2%) and CR
conditions (0.5%), suggesting that reducing the
quantity of the competitive Sir2p inhibitor NADH
might also contribute to the CR-induced increase in
yeast longevity (26). Interestingly, Hst2, a Sir2
homolog that contributes to rDNA stability, is
another mediator of CR-induced life span extension
in yeast, indicating that other Sir2 homologs could
regulate longevity under CR conditions (29). Taken
together, these data suggest that Sir2 requires NAD
to deacetylate proteins, and manipulations of the
salvage pathway that converge on enhanced NAD
availability induce Sir2-dependent life span
Figure 2. A: yeast and mammalian NAD
salvage pathways. Yeast (red) Sir2p utilizes the cofactor NAD
to deacetylate proteins and in this
reaction produces nicotinamide (NAM) and O-acetyl-ADP-ribose (O-AA-ribose). NAM is deaminated by the nicotinamidase PNC1 to
form nicotinic acid (NA). NA will give rise successively to nicotinate mononucleotide (NAMN), nicotinate adenine dinucleotide (NAAD)
and nicotinamide adenine dinucleotide (NAD
) by the enzymes nicotinate phosphoribosyl-transferase (NPT1), nicotinate mononucleotide
adenyltransferase (NMAT), and nicotinamide adenine dinucleotide (NAD) synthetase (QNS), respectively (red arrows). In mammals
(blue) NAM is recycled to NAD
in two steps through the formation of nicotinamide mononucleotide (NMN) by means of the NAM
phosphoribosyltransferase NamPT (PBEF/visfatin) and nicotinamide mononucleotide adenyltransferase (NMNAT) (blue arrows). B:
SIRT1 protective functions in metabolism and diseases. SIRT1 can be regulated positively by CR and SIRT-activating compounds and
negatively by SIRT inhibitors. SIRT1 activation induces survival of cardiomyocytes, protects neurons from cell death, and favors insulin
secretion by repressing the uncoupling protein 2 (UCP2). SIRT1 decreases white adipocyte tissue formation through repression of PPARc
and promotes gluconeogenesis in response to fasting through PGC-1a, and stimulates mitochondrial biogenesis in the brown adipose tissue
(BAT) and the muscle through activation of PGC-1a.NA5nicotinic acid; NAD
5nicotinamide adenine dinucleotide; NAM5
nicotinamide; NMN5nicotinamide mononucleotide; PNC15 pyrazinamidase/Nicotinamidase 1; NamPT15nicotinamide phosphoribosyl
transferase 1; PBEF5pre-B cell enhancing factor; NPT15nicotinate phosphoribosyl transferase 1; NMAT1/25nicotinate mononucleotide
adenyltransferase 1/2; QNS5NAD synthetase; NMNAT15nicotinamide mononucleotide adenyltransferase; O-AA-ribose5O-acetyl-
ADP-ribose; UCP-25uncoupling protein 2; NFkB5nuclear factor kappa B; PPARc5peroxisome proliferator-activated receptor gamma;
PGC-1a5peroxisome proliferator-activated receptor gamma coactivator-1 alpha; BAT5brown adipose tissue; SIRT15sirtuin 1.
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extension in the budding yeast (24). Under CR other
pathways, such as a decrease in the competitive Sir2
inhibitor NADH (by increasing Nde activity) or the
noncompetitive Sir2 inhibitor NAM (by increasing
PNC1 activity), could contribute to control Sir2
activity. Furthermore, a Sir2-independent CR-
mediated life span extension mechanism exists that
is mediated via Hst2.
Three important factors are thought to participate
in Sir2-mediated longevity. First, one of the con-
sequences of increased Sir2 activity is the significant
reduction in the number of extrachromosomal
circular DNA repeats (ERCs) (11), whose accumu-
lation is deleterious to yeast and accelerates ageing
(30), probably through sequestration of transcrip-
tion factors essential for their replication. The
second mechanism that contributes to the yeast life
span acts via the fermentable growth medium-
induced (FGM) pathway. Under fermentation con-
ditions, glucose and other nutrients activate a yet
unknown pathway that activates Sch9, a kinase
related to the mammalian protein kinase B (PKB)/
AKT, a negative regulator of the stress resistance
genes. This results in the subsequent accumulation
of reactive oxygen species (ROS), thereby accelerat-
ing ageing (31). Interestingly, mutation of Sch9
yields a substantial increase in yeast life span
probably through increasing stress resistance
(32,33) (Figure 1). It is at present not known
whether this pathway is similar to Daf-16/FOXO
signaling, which participates in the control of ageing
in other organisms, and whether Sch9 mutations
affect ERCs formation. Finally, yeast evolving in a
high osmolarity live much longer than those in a
balanced osmolarity medium. A high osmolarity
shock activates a subset of osmotic responsive genes
that change the metabolism to favor glycerol
biosynthesis thereby generating more NAD
, the
Sir2 cofactor necessary to mediate longevity (34).
Studies in Caenorhabditis elegans and Drosophila
In C. elegans, the yeast Sir2p ortholog, Sir2.1,
extends life span through the forkhead transcription
factor Daf-16 (homolog of mammalian FOXO)
signaling pathway. Loss of function studies of
components of the Daf-2 (homolog of the mamma-
lian insulin receptor) signaling pathway, such as in
Daf-2 or the homolog of the mammalian phospha-
tidyl-inositol-3 kinase (PI3K), AGE-1, extend the
worm’s life span (Figure 1) (35,36). This pathway
controls the entry of worms in ‘dauer’, a larval
developmental state of growth arrest that is induced
upon food limitation, and which is a feature of young
larvae before reproductive maturation (reviewed in
(37)). Activation of Daf-2/AGE-1 signaling results in
the phosphorylation of AKT kinase that sequesters
Daf-16 in the cytoplasm resulting in its inactivation
(38,39). The long-lived mutants require the entry of
Daf-16 into the nucleus to activate target genes
necessary for dauer formation. Duplication of
chromosomal regions containing Sir2.1 in the
nematode extends life span by up to 50%.
Interestingly, the Sir2.1-duplicated strains carrying
a mutated Daf-16 displayed the same decreased life
span as observed in Daf-16 mutants, proving that
Sir2.1 acts upstream of Daf-16. In addition, Sir2.1-
duplicated strains do not further extend life span of
Daf-2 mutants indicating that extra copies of Sir2.1
promote longevity through the Daf-2/Daf-16 signal-
ing pathway (12). A direct interaction between
Sir2.1 and Daf-16, facilitated by the nematode 14-
3-3 protein, furthermore suggests that these three
proteins physically interact (40,41).
Also dSir2, the drosophila Sir2 ortholog, controls
life span extension under CR conditions, and
mutants that remove or decrease dSir2 levels are
no longer able to promote the CR-mediated long-
evity. The dSir2-mediated life extension seems to
work in the same pathway as the Rpd3 histone
deacetylase, since Rpd3 is thought to negatively
regulate dSir2, and CR induces a decrease in Rpd3
expression (13). Moreover, long-lived Rpd3 mutant
flies display high dSir2 expression levels (42).
Interestingly, flies deficient in the homolog of the
mammalian insulin receptor substrate (IRS) protein,
CHICO, show a significant increase in life span
(43). Recent studies showed that activation of
dFOXO in the fly brain and/or fat body extends life
span and inhibits the endogenous insulin-dependent
signaling in the fat body (44,45). Although both CR
and downregulation of the insulin pathway partici-
pate in life span extension, it is still not proven that
the two pathways work in concert and converge on
dFOXO to promote longevity in drosophila.
Studies in mammals
The mammalian NAD
salvage pathway is different
from the yeast pathway. NAM is directly trans-
formed to nicotinamide mononucleotide (NMN) by
the enzyme nicotinamide phosphoribosyltransferase
(NamPT) (46), which then yields NAD
the action of nicotinamide mononucleotide adenyl
transferase (NMNAT) (Figure 2A). NamPT was
found to be the equivalent of the pre-B-cell-enhancing
factor (PBEF), a protein that stimulated B-cell colony
formation (47), and visfatin, an adipokine expressed in
visceral fat with glucose-lowering properties (48).
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Although some evidence exists that NamPT activates
SIRT1 transcriptional activity (27), NamPT over-
expression in mice would be necessary to determine
whether NamPT is the functional homolog of yeast
PNC1 in life span extension.
The role of the insulin-signaling pathway in the
determination of mammalian life span has been
studied in insulin-like growth factor receptor (IGF-
R)-deficient mice. Although IGF-R
mice die
shortly after birth, IGF-R
animals are viable,
display reduced IGF-R levels, and live longer than
littermates. IGF-R
animals exhibit low
AKT kinase activity suggesting an increase in FOXO
activity, reproducing features of the Daf-16-
mediated longevity pathway in C. elegans. In addi-
tion, IGF-R
mice are more resistant to oxidative
stress. This phenotype was, however, gender-depen-
dent since only IGF-R
female mice displayed a
significant increase in life span, whereas IGF-R
males showed a modest, non-significant increase in
longevity (49). Mice in which the insulin receptor was
specifically inactivated in the adipose tissue also live
longer and are protected against age-related obesity
and its subsequent metabolic abnormalities (50).
In mammals, SIRT1 has been linked with meta-
bolic control. The importance of SIRT1 in the
nutrient control of glucose homeostasis was demon-
strated to involve the modulation of the acetylation
status and hence the stimulation of the activity of the
metabolic coregulator peroxisome proliferator-acti-
vated receptor gamma (PPARc) coactivator-1a
(PGC-1a). In the liver, SIRT1 in a complex
including the hepatocyte nuclear factor-4 (HNF-4)
deacetylates and activates PGC-1a, promoting glu-
coneogenesis following fasting (51). SIRT1 also
functions together with PGC-1a, beyond the liver.
Indeed, activated PGC-1a promotes mitochondrial
function in the skeletal muscle and the brown
adipose tissue, leading to enhanced energy expendi-
ture, increased exercise performance, and protection
from diet-induced insulin resistance and hepatostea-
tosis (16,52). Although these studies suggest that
PGC-1a is a privileged partner for SIRT1, other
signaling pathways are also solicited (Figure 2B).
For instance, in white adipose tissue, where the
activation of SIRT1 by resveratrol decreased fat
accumulation and its inhibition resulted in triglycer-
ide accumulation, it was suggested that SIRT1
activation repressed the nuclear receptor PPARc
(53), an effect that, in addition to SIRT1, also
involved the nuclear receptor corepressor (NcoR)
and silencing mediator SMRT (54). A recent study
also emphasized the role of the endothelial nitric
oxide synthase (eNOS) signaling in CR-mediated
increases in mitochondrial biogenesis and life span in
mice. CR animals showed an increase in mitochon-
drial biogenesis that correlated with an increase in
the expression of SIRT1, PGC-1a and eNOS in
various tissues. Interestingly, these effects were
completely inhibited in eNOS
animals in which
life span was significantly reduced (55).
SIRT1 has also been demonstrated to augment
insulin secretion in response to glucose in pancreatic
b-cells of b-cell-specific SIRT1-overexpressing
(BESTO) transgenic mice (56). This response was
accompanied by a decrease in the expression of the
uncoupling protein-2 (UCP-2) that could increase
ATP production in the b-cells of BESTO transgenic
mice. UCPs uncouple oxygen consumption from
ATP generation by allowing leakage of protons (H
thereby participating in the reduction of ROS
generation. The SIRT1-mediated decrease in
UCP-2 expression impedes H
‘leakage’ and allows
a more efficient coupling of electron transport with
the ATP production. Likewise, UCP-2
mice also
exhibit a similar phenotype with improved glucose
tolerance and enhanced insulin secretion (57).
SIRT1 achieves this effect on UCP-2 expression by
directly binding to and repressing UCP-2 gene
transcription in pancreatic b-cells (58). SIRT1 can
also form a complex with FOXO1 and the promye-
locytic leukemia protein PML to activate two insulin
transcription factors, NeuroD and MafA, which may
protect the pancreatic b-cell pathway from oxidative
damage (59). An approach to increase SIRT1
dosage in pancreatic b-cells could hence be of
potential interest to maintain a healthy b-cell
function in diabetic patients.
SIRT1 and SIRT3 deacetylate acetyl coenzyme A
synthetase (AceCS). AceCS catalyzes the formation
of acetyl-CoA from acetate, coenzyme A, and ATP.
Whereas SIRT1 has been shown to deacetylate the
cytoplasmic AceCS1, whose activity controls acetyl-
CoA levels in the cytoplasm for fatty acid synthesis,
SIRT3 deacetylates the mitochondrial AceCS2
which regulates acetyl-CoA-requiring pathways in
the mitochondria such as the tricarboxylic acid cycle
(60). Since acetate metabolism is impaired in
diabetes and ageing, it is legitimate to speculate on
the potential role of SIRT1 and SIRT3 in the
pathophysiology of these diseases through the
regulation of AceCS1 and AceCS2. Further valida-
tion of these observations in in vivo models is still
A central role for SIRT1 in disease of the central
nervous system has also been highlighted in animal
models. In the Wallerian degeneration slow (Wld
mouse model, SIRT1 activation protects axons
against neuronal injury. This Wld
mouse bears
in fact a dominant mutation producing an
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overexpressed chimeric Wld
protein composed of
the ubiquitin assembly protein Ufd2a and the NAD
salvage pathway enzyme NMNAT1. Decreasing
SIRT1 activity reduces the axonal protection origin-
ally observed, whereas SIRT1 activation by resver-
atrol decreases the axonal degeneration after
neuronal injury. This suggests that the neuroprotec-
tion in the Wld
mouse model is achieved by
increasing the neuronal NAD
reserve and/or
SIRT1 activity (61). Furthermore it has been
reported that the SIRT1 agonist resveratrol protects
C. elegans neurons expressing a fragment of the
Huntington disease-associated protein huntingtin
and mammalian neurons from mutant polygluta-
mine cytotoxicity in a HdhQ111 knock-in mouse
model of Huntington disease (62). In addition,
SIRT1 activation significantly decreases neuronal
cell death induced by amyloid-beta (Ab) peptides
through inhibition of NFkB signaling (63). Specific
brain hSIRT1 overexpression in transgenic animals
induces a significant increase in the a-secretase
activity, an enzyme that cleaves the amyloid pre-
cursor peptide (APP) within the Ab peptide,
favoring thereby the nonamyloidogenic pathway of
the APP processing (64). In addition, a recent study
demonstrated the protective effect of CR against
Alzheimer’s disease-type brain amyloidosis in mon-
keys. Monkeys maintained on CR diet had signifi-
cantly reduced contents of Ab peptides in the
temporal cortex that correlated with enhanced
SIRT1 concentrations as compared to monkeys
under normal diet (65). From these studies, it
became clear that pharmacological strategies aiming
at activating SIRT1 would impede Ab peptide
deposition in the brain and prevent the development
of Alzheimer’s disease.
From the animal studies discussed above, it was
suggested that SIRT1 could contribute to the
pathogenesis of some complex diseases. SIRT1
could hence be considered as a serious candidate
target for therapeutic interventions in metabolic and
neurodegenerative disorders. In line with this
hypothesis, genetic variants (SNPs) in the human
SIRT1 gene have been shown to be tightly asso-
ciated with energy expenditure (52). We predict that
future human studies will link SIRT1 even more
tightly with metabolic and neurodegenerative dis-
eases, stimulating the development of therapeutics
for their treatment.
Emerging functions for the other sirtuins
The tubulin-deacetylase protein SIRT2 (66) has
been demonstrated to be downregulated in human
gliomas, which are amongst the most frequent
malignant brain tumors (67). The SIRT2 gene maps
at chromosome 19q13.2, a region frequently deleted
in human gliomas. Furthermore, ectopic expression
of SIRT2 in a glioma cell line decreased colony
formation suggesting a potential tumor suppressor
role of SIRT2. This could be explained by the fact
that SIRT2 plays an important role in the control of
mitotic exit in the cell cycle where increased SIRT2
activity severely delays cell cycle progression through
mitosis (68), but also by the fact that SIRT2 acts as a
mitotic checkpoint protein that prevents chromoso-
mal instability and the formation of hyperploid cells
in the early metaphase (69). Further studies such as
SIRT2 brain-specific inactivation in genetically
engineered mice should bring an insight into the
mechanism and the role of SIRT2 in the pathophy-
siology of this aggressive type of brain cancer. Very
recently, SIRT2 was described as an oligodendro-
glial cytoplasmic protein, localized to the outer and
juxtanodal loops in the myelin sheath, that decreases
cell differentiation through a-tubulin deacetylation
suggesting a potential implication in myelinogenesis
The mitochondrial SIRT3 deacetylase (71,72) has
been linked with adaptative thermogenesis. SIRT3
expression is induced in mice in both white and
brown adipose tissue (BAT) by CR and in BAT
upon cold exposure. SIRT3 furthermore activates
known mitochondrial genes such as PGC-1a and
UCP-1 suggesting an important role of SIRT3 in
thermogenesis (73). In addition, the SIRT3 gene has
been linked to longevity. In fact, the genotype
defined by the G477T polymorphism in exon 3,
was associated with survivorship (74). Furthermore,
a VNTR (a 72-bp repeat core) polymorphism in
intron 5 of the SIRT3 gene, that acts as an allele-
specific enhancer activity on a reporter gene, was
associated with longevity in male subjects (75). This
VNTR might represent the functional variant that
accounts for the association between the silent
marker G477T and longevity in old male subjects.
Although the mitochondrial expressed protein
SIRT4 has a conserved sirtuin domain, it seems
not to possess in vitro deacetylase activity (76).
However, SIRT4 ADP-ribosylates and inhibits the
mitochondrial glutamate dehydrogenase (GDH).
GDH controls amino acid-stimulated insulin secre-
tion by regulating glutamine and glutamate oxidative
Sirtuins and metabolism 341
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metabolism. Inhibition of GDH activity by SIRT4
decreases insulin secretion in mouse pancreatic b-
cells in response to amino acids (76). Interestingly,
the two different sirtuins SIRT1 and SIRT4 seem to
work in opposite directions to control insulin
secretion. Furthermore SIRT4 expression is down-
regulated in response to CR in b-cells, which is
opposite to the regulation of SIRT1 during CR.
More studies are still needed to see whether SIRT4
(and SIRT1) can be integrated in the pathophysiol-
ogy of type 1 and 2 diabetes that both are
characterized by alterations in insulin secretion.
Whereas little is known about the activity of SIRT5,
SIRT6 is suggested to control genomic DNA
stability and DNA repair. SIRT6
mice die
prematurely and display severe defects including
important lymphopenia, loss of subcutaneous fat,
decreased bone mineral density, metabolic defects
with a profound imbalance in glucose homeostasis
and decreased IGF-1 levels. The phenotype of the
mice mimics multiple pathologies found
in elderly humans (77). Although SIRT6 was
originally described as an exclusive ADP-ribosyl-
transferase (78), it was recently demonstrated that
SIRT6 deacetylates histones and the DNA repair
enzyme, DNA polymerase b (polb) in vitro (77). An
effect on DNA repair was hence proposed to explain
the phenotype in SIRT6
mice, and it was
suggested that SIRT6 could play an essential role
in maintaining organ integrity as animals age.
SIRT7 is the only sirtuin to be localized in the
nucleolus and is a component of the RNA poly-
merase I (Pol I) transcriptional machinery. SIRT7
interacts with RNA Pol I and histones, and positively
regulates the transcription of rDNA during tran-
scriptional elongation, which accounts for about
60% of total transcription in metabolically active
cells in mammals (79). SIRT7 overexpression
increases Pol I-mediated transcription in a NAD
dependent manner, whereas SIRT7 inhibition
induces a decrease in the transcription of Pol I and
its association with rDNA (80). Depletion of
SIRT7 stopped cell proliferation and triggered
apoptosis. It was suggested that SIRT7 may regu-
late rDNA transcription by sensing cellular NAD
levels and that diet-induced changes in NAD
NADH ratio might modulate SIRT7 to link the
cellular energy status with rRNA synthesis and
Table I. Expression pattern, cellular distribution, and function of sirtuin deacetylases. Sirtuins are differentially expressed in different
organs based on their transcripts.
Expression levels
target genes
Potential link
with diseasesHigh Low
SIRT1 Br, Te, Sk,
Ki (+++),
Th, Ut (++)
Li, Sp, He Lu,
Ov, BM
Nuclear p53,Ku70, NFkB,
PGC-1a, MEF2D,
MyoD, PPARc,
FOXO, p300,
AceCS1, tat
ageing, obesity, Insulin
resistance, inflammation,
diabetes, heart failure, axonal
degeneration, AIDS
SIRT2 Br, Sk (+++), Li,
Te, Ki, He (++)
Th, Lu, BM, Ut,
Ov, Sp
Cytoplasmic a-tubulin downregulated in human gliomas
SIRT3 Ov (+++), most
other organs (++)
Mitochondrial PGC-1a, AceCS2 adaptive thermogenesis,
overexpressed in node-positive
breast cancer
SIRT4 Br, Te, He,
Lu (+++), Li,
Sk, Ki, Th, Ut,
Ov (++)
BM, Sp Mitochondrial Glutamate
inhibits amino acid-stimulated
insulin secretion
SIRT5 Br, Te, Sk, Ki, He
(+++) Li, Ov, Lu,
Th, Ut, BM (++)
Sp Mitochondrial unknown unknown
SIRT6 Fetal Br (+++), Br,
Li, Te, Sk, Ki, He,
Ov (++)
Sp, Th, Ut, BM, Lu Nuclear DNA polb age-related diseases
SIRT7 Br, Te, Ki Lower in other organs Nuclear RNA polymerase
Pol I
highly expressed in thyroid
cancers, overexpressed in node-
positive breast cancer
Sp, Te, Li, Ki,
Pa (proteins)
Br, Sk, He (proteins)
Abbreviations: Br5Brain; He5heart; Ki5kidney; Sk5skeletal muscle; Li
5liver; Te5testis; Lu5lung; Sp5spleen; Ov5ovary; Ut5uterus;
BM5bone marrow; Th5thyroid.
342 N. Dali-Youcef et al.
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ribosome production. It is worth noting that SIRT1
negatively regulates RNA Pol I through deacetyla-
tion of TAFI68 (81), which goes opposite of SIRT7
Concluding remarks
In the last decade, sirtuin biology has come a long
way from their original description as yeast NAD
dependent class III HDACs, that control yeast life
span. In mammals, seven orthologs of Sir2 have
been identified, SIRT1 to 7, and the exact biological
function of most of these sirtuins still remains only
partially characterized. Of particular interest is the
fact that SIRT1 not only deacetylates histones to
mediate gene silencing, but is able to interact with
and deacetylate some well known transcriptional
regulators thereby modulating specifically various
biological processes. Hence modulating the expres-
sion of SIRT1 or its activity, by using sirtuin-
activating compounds such as resveratrol, will have
pleiotropic effects. SIRT1 activation reduces fat
accumulation and adipocyte differentiation through
repression of the activity of the adipogenic nuclear
receptor PPARc. SIRT1 also promotes mitochon-
drial function and energy expenditure and conse-
quently protects mice from diet-induced obesity,
through deacetylation and subsequent activation of
PGC-1a in the skeletal muscle and in the brown
adipose tissue. The SIRT1/PGC-1a interaction is
also important in the liver, where SIRT1 activation
upon fasting induces gluconeogenesis and prevents
against hepatosteatosis. In addition, SIRT1 signifi-
cantly enhances insulin secretion in the pancreatic b-
cells. In combination, these studies illustrate that
SIRT1 is a major modulator of metabolism. SIRT1
activation also seems to be endowed with neuropro-
tective activities as suggested from the study of
models of Huntington or Alzheimer’s disease.
Furthermore, other sirtuins might play important
roles in some diseases, as illustrated by SIRT2,
which is downregulated in human gliomas.
Obviously, more studies, in animal models and
humans, are still needed to define the exact role of
sirtuins in the pathophysiology of human diseases. It
can, however, be predicted that therapeutic inter-
ventions aiming at activating or blocking sirtuins,
depending on the context, will one day become
helpful in the treatment of human diseases.
We thank greatly members of the Auwerx laboratory
for critical reading of the manuscript and helpful
discussions. This work was supported by grants of
the Centre National de la Recherche Scientifique
(CNRS); Institut National pour la Science et la
Recherche Me´dicale (INSERM); National Institutes
of Health (NIH); the European Union (EU) and the
Hoˆpitaux Universitaires de Strasbourg.
1. Chen H, Lin RJ, Xie W, Wilpitz D, Evans RM. Regulation of
hormone-induced histone hyperacetylation and gene activa-
tion via acetylation of an acetylase. Cell. 1999;98:675–86.
2. Courey AJ, Jia S. Transcriptional repression: the long and the
short of it. Genes Dev. 2001;15:2786–96.
3. Imai S, Johnson FB, Marciniak RA, McVey M, Park PU,
Guarente L. Sir2: an NAD-dependent histone deacetylase
that connects chromatin silencing, metabolism, and aging.
Cold Spring Harb Symp Quant Biol. 2000;65:297–302.
4. Imai S, Armstrong CM, Kaeberlein M, Guarente L.
Transcriptional silencing and longevity protein Sir2 is an
NAD-dependent histone deacetylase. Nature. 2000;403:
5. Lin SJ, Guarente L. Nicotinamide adenine dinucleotide, a
metabolic regulator of transcription, longevity and disease.
Curr Opin Cell Biol. 2003;15:241–6.
6. Hoppe GJ, Tanny JC, Rudner AD, Gerber SA, Danaie S,
Gygi SP, et al. Steps in assembly of silent chromatin in yeast:
Sir3-independent binding of a Sir2/Sir4 complex to silencers
and role for Sir2-dependent deacetylation. Mol Cell Biol.
7. Ivy JM, Klar AJ, Hicks JB. Cloning and characterization of
four SIR genes of Saccharomyces cerevisiae. Mol Cell Biol.
8. Rine J, Herskowitz I. Four genes responsible for a position
effect on expression from HML and HMR in Saccharomyces
cerevisiae. Genetics. 1987;116:9–22.
9. Chen L, Widom J. Mechanism of transcriptional silencing in
yeast. Cell. 2005;120:37–48.
10. Braunstein M, Rose AB, Holmes SG, Allis CD, Broach JR.
Transcriptional silencing in yeast is associated with reduced
nucleosome acetylation. Genes Dev. 1993;7:592–604.
11. Kaeberlein M, McVey M, Guarente L. The SIR2/3/4
complex and SIR2 alone promote longevity in
Saccharomyces cerevisiae by two different mechanisms.
Genes Dev. 1999;13:2570–80.
12. Tissenbaum HA, Guarente L. Increased dosage of a sir-2
gene extends lifespan in Caenorhabditis elegans. Nature.
13. Rogina B, Helfand SL. Sir2 mediates longevity in the fly
through a pathway related to calorie restriction. Proc Natl
Acad Sci U S A. 2004;101:15998–6003.
14. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW,
Lavu S, Wood JG, et al. Small molecule activators of sirtuins
extend Saccharomyces cerevisiae lifespan. Nature. 2003;425:
15. Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL,
Tatar M, et al. Sirtuin activators mimic caloric restriction
and delay ageing in metazoans. Nature. 2004;430:686–9.
16. Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C,
Kalra A, et al. Resveratrol improves health and survival of
mice on a high-calorie diet. Nature. 2006;444:337–42.
17. Bitterman KJ, Anderson RM, Cohen HY, Latorre-Esteves M,
Sinclair DA. Inhibition of silencing and accelerated aging by
nicotinamide, a putative negative regulator of yeast sir2 and
human SIRT1. J Biol Chem. 2002;277:45099–107.
Sirtuins and metabolism 343
Ann Med Downloaded from by Universite Louis Pasteur on 06/17/10
For personal use only.
18. Bordone L, Guarente L. Calorie restriction, SIRT1 and
metabolism: understanding longevity. Nat Rev Mol Cell Biol.
19. Lin SJ, Defossez PA, Guarente L. Requirement of NAD and
SIR2 for life-span extension by calorie restriction in
Saccharomyces cerevisiae. Science. 2000;289:2126–8.
20. Lin SJ, Kaeberlein M, Andalis AA, Sturtz LA, Defossez PA,
Culotta VC, et al. Calorie restriction extends Saccharomyces
cerevisiae lifespan by increasing respiration. Nature.
21. Bakker BM, Overkamp KM, van Maris AJ, Kotter P,
Luttik MA, van Dijken JP, et al. Stoichiometry and
compartmentation of NADH metabolism in Saccharomyces
cerevisiae. FEMS Microbiol Rev. 2001;25:15–37.
22. Tanner KG, Landry J, Sternglanz R, Denu JM. Silent
information regulator 2 family of NAD-dependent histone/
protein deacetylases generates a unique product, 1-O-acetyl-
ADP-ribose. Proc Natl Acad Sci U S A. 2000;97:14178–82.
23. Liou GG, Tanny JC, Kruger RG, Walz T, Moazed D.
Assembly of the SIR complex and its regulation by O-acetyl-
ADP-ribose, a product of NAD-dependent histone deacetyla-
tion. Cell. 2005;121:515–27.
24. Anderson RM, Bitterman KJ, Wood JG, Medvedik O,
Cohen H, Lin SS, et al. Manipulation of a nuclear NAD+
salvage pathway delays aging without altering steady-state
NAD+ levels. J Biol Chem. 2002;277:18881–90.
25. Anderson RM, Bitterman KJ, Wood JG, Medvedik O,
Sinclair DA. Nicotinamide and PNC1 govern lifespan
extension by calorie restriction in Saccharomyces cerevisiae.
Nature. 2003;423:181–5.
26. Lin SJ, Ford E, Haigis M, Liszt G, Guarente L. Calorie
restriction extends yeast life span by lowering the level of
NADH. Genes Dev. 2004;18:12–6.
27. Revollo JR, Grimm AA, Imai S. The NAD biosynthesis
pathway mediated by nicotinamide phosphoribosyltransferase
regulates Sir2 activity in mammalian cells. J Biol Chem.
28. Sandmeier JJ, Celic I, Boeke JD, Smith JS. Telomeric and
rDNA silencing in Saccharomyces cerevisiae are dependent
on a nuclear NAD(+) salvage pathway. Genetics. 2002;160:
29. Lamming DW, Latorre-Esteves M, Medvedik O, Wong SN,
Tsang FA, Wang C, et al. HST2 mediates SIR2-independent
life-span extension by calorie restriction. Science. 2005;309:
30. Sinclair DA, Guarente L. Extrachromosomal rDNA circles—
a cause of aging in yeast. Cell. 1997;91:1033–42.
31. Longo VD. The Ras and Sch9 pathways regulate stress
resistance and longevity. Exp Gerontol. 2003;38:807–11.
32. Fabrizio P, Pozza F, Pletcher SD, Gendron CM, Longo VD.
Regulation of longevity and stress resistance by Sch9 in yeast.
Science. 2001;292:288–90.
33. Fabrizio P, Liou LL, Moy VN, Diaspro A,
SelverstoneValentine J, Gralla EB, et al. SOD2 functions
downstream of Sch9 to extend longevity in yeast. Genetics.
34. Kaeberlein M, Andalis AA, Fink GR, Guarente L. High
osmolarity extends life span in Saccharomyces cerevisiae by a
mechanism related to calorie restriction. Mol Cell Biol.
35. Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G. daf-2, an
insulin receptor-like gene that regulates longevity and
diapause in Caenorhabditis elegans. Science. 1997;277:
36. Morris JZ, Tissenbaum HA, Ruvkun G. A phosphatidylino-
sitol-3-OH kinase family member regulating longevity and
diapause in Caenorhabditis elegans. Nature. 1996;382:
37. Burnell AM, Houthoofd K, O’Hanlon K, Vanfleteren JR.
Alternate metabolism during the dauer stage of the nematode
Caenorhabditis elegans. Exp Gerontol. 2005;40:850–6.
38. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L,
Tissenbaum HA, et al. The Fork head transcription factor
DAF-16 transduces insulin-like metabolic and longevity
signals in C. elegans. Nature. 1997;389:994–9.
39. Lin K, Dorman JB, Rodan A, Kenyon C. daf-16: An HNF-3/
forkhead family member that can function to double the life-
span of Caenorhabditis elegans. Science. 1997;278:1319–22.
40. Berdichevsky A, Viswanathan M, Horvitz HR, Guarente L.
C. elegans SIR-2.1 interacts with 14-3-3 proteins to activate
DAF-16 and extend life span. Cell. 2006;125:1165–77.
41. Wang Y, Oh SW, Deplancke B, Luo J, Walhout AJ,
Tissenbaum HA. C. elegans 14-3-3 proteins regulate life
span and interact with SIR-2.1 and DAF-16/FOXO. Mech
Ageing Dev. 2006;127:741–7.
42. Rogina B, Helfand SL, Frankel S. Longevity regulation by
Drosophila Rpd3 deacetylase and caloric restriction. Science.
43. Clancy DJ, Gems D, Harshman LG, Oldham S, Stocker H,
Hafen E, et al. Extension of life-span by loss of CHICO, a
Drosophila insulin receptor substrate protein. Science.
44. Hwangbo DS, Gershman B, Tu MP, Palmer M, Tatar M.
Drosophila dFOXO controls lifespan and regulates insulin
signalling in brain and fat body. Nature. 2004;429:562–6.
45. Giannakou ME, Goss M, Junger MA, Hafen E, Leevers SJ,
Partridge L. Long-lived Drosophila with overexpressed
dFOXO in adult fat body. Science. 2004;305:361.
46. Rongvaux A, Shea RJ, Mulks MH, Gigot D, Urbain J, Leo O,
et al. Pre-B-cell colony-enhancing factor, whose expression is
up-regulated in activated lymphocytes, is a nicotinamide
phosphoribosyltransferase, a cytosolic enzyme involved in
NAD biosynthesis. Eur J Immunol. 2002;32:3225–34.
47. Samal B, Sun Y, Stearns G, Xie C, Suggs S, McNiece I.
Cloning and characterization of the cDNA encoding a novel
human pre-B-cell colony-enhancing factor. Mol Cell Biol.
48. Fukuhara A, Matsuda M, Nishizawa M, Segawa K,
Tanaka M, Kishimoto K, et al. Visfatin: a protein secreted
by visceral fat that mimics the effects of insulin. Science.
49. Holzenberger M, Dupont J, Ducos B, Leneuve P, Geloen A,
Even PC, et al. IGF-1 receptor regulates lifespan and
resistance to oxidative stress in mice. Nature. 2003;421:
50. Bluher M, Kahn BB, Kahn CR. Extended longevity in mice
lacking the insulin receptor in adipose tissue. Science.
51. Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM,
Puigserver P. Nutrient control of glucose homeostasis
through a complex of PGC-1alpha and SIRT1. Nature.
52. Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H,
Lerin C, Daussin F, et al. Resveratrol improves mitochondrial
function and protects against metabolic disease by activating
SIRT1 and PGC-1alpha. Cell. 2006;127:1109–22.
53. Picard F, Auwerx J. PPAR(gamma) and glucose homeostasis.
Annu Rev Nutr. 2002;22:167–97.
54. Picard F, Kurtev M, Chung N, Topark-Ngarm A,
Senawong T, Machado De Oliveira R, et al. Sirt1 promotes
fat mobilization in white adipocytes by repressing PPAR-
gamma. Nature. 2004;429:771–6.
344 N. Dali-Youcef et al.
Ann Med Downloaded from by Universite Louis Pasteur on 06/17/10
For personal use only.
55. Nisoli E, Tonello C, Cardile A, Cozzi V, Bracale R,
Tedesco L, et al. Calorie restriction promotes mitochondrial
biogenesis by inducing the expression of eNOS. Science.
56. Moynihan KA, Grimm AA, Plueger MM, Bernal-Mizrachi E,
Ford E, Cras-Meneur C, et al. Increased dosage of
mammalian Sir2 in pancreatic beta cells enhances glucose-
stimulated insulin secretion in mice. Cell Metab.
57. Zhang CY, Baffy G, Perret P, Krauss S, Peroni O, Grujic D,
et al. Uncoupling protein-2 negatively regulates insulin
secretion and is a major link between obesity, beta cell
dysfunction, and type 2 diabetes. Cell. 2001;105:745–55.
58. Bordone L, Motta MC, Picard F, Robinson A, Jhala US,
Apfeld J, et al. Sirt1 regulates insulin secretion by repressing
UCP2 in pancreatic beta cells. PLoS Biol. 2006;4:e31.
59. Kitamura YI, Kitamura T, Kruse JP, Raum JC, Stein R,
Gu W, et al. FoxO1 protects against pancreatic beta cell
failure through NeuroD and MafA induction. Cell Metab.
60. Hallows WC, Lee S, Denu JM. Sirtuins deacetylate and
activate mammalian acetyl-CoA synthetases. Proc Natl Acad
Sci U S A. 2006;103:10230–5.
61. Araki T, Sasaki Y, Milbrandt J. Increased nuclear NAD
biosynthesis and SIRT1 activation prevent axonal degenera-
tion. Science. 2004;305:1010–3.
62. Parker JA, Arango M, Abderrahmane S, Lambert E,
Tourette C, Catoire H, et al. Resveratrol rescues mutant
polyglutamine cytotoxicity in nematode and mammalian
neurons. Nat Genet. 2005;37:349–50.
63. Chen J, Zhou Y, Mueller-Steiner S, Chen LF, Kwon H, Yi S,
et al. SIRT1 protects against microglia-dependent amyloid-
beta toxicity through inhibiting NF-kappaB signaling. J Biol
Chem. 2005;280:40364–74.
64. Qin W, Yang T, Ho L, Zhao Z, Wang J, Chen L, et al.
Neuronal SIRT1 activation as a novel mechanism underlying
the prevention of Alzheimer disease amyloid neuropathology
by calorie restriction. J Biol Chem. 2006;281:21745–54.
65. Qin W, Chachich M, Lane M, Roth G, Bryant M, de Cabo R,
et al. Calorie restriction attenuates Alzheimer’s disease type
brain amyloidosis in Squirrel monkeys (Saimiri sciureus). J
Alzheimers Dis. 2006;10:417–22.
66. North BJ, Marshall BL, Borra MT, Denu JM, Verdin E. The
human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin
deacetylase. Mol Cell. 2003;11:437–44.
67. Hiratsuka M, Inoue T, Toda T, Kimura N, Shirayoshi Y,
Kamitani H, et al. Proteomics-based identification of
differentially expressed genes in human gliomas: down-
regulation of SIRT2 gene. Biochem Biophys Res Commun.
68. Dryden SC, Nahhas FA, Nowak JE, Goustin AS,
Tainsky MA. Role for human SIRT2 NAD-dependent
deacetylase activity in control of mitotic exit in the cell cycle.
Mol Cell Biol. 2003;23:3173–85.
69. Inoue T, Hiratsuka M, Osaki M, Yamada H, Kishimoto I,
Yamaguchi S, et al. SIRT2, a tubulin deacetylase, acts to
block the entry to chromosome condensation in response to
mitotic stress. Oncogene. 2007;26:945–57.
70. Li W, Zhang B, Tang J, Cao Q, Wu Y, Wu C, et al. Sirtuin 2,
a mammalian homolog of yeast silent information regulator-2
longevity regulator, is an oligodendroglial protein that
decelerates cell differentiation through deacetylating alpha-
tubulin. J Neurosci. 2007;27:2606–16.
71. Onyango P, Celic I, McCaffery JM, Boeke JD, Feinberg AP.
SIRT3, a human SIR2 homologue, is an NAD-dependent
deacetylase localized to mitochondria. Proc Natl Acad Sci U
S A. 2002;99:13653–8.
72. Schwer B, North BJ, Frye RA, Ott M, Verdin E. The human
silent information regulator (Sir)2 homologue hSIRT3 is a
mitochondrial nicotinamide adenine dinucleotide-dependent
deacetylase. J Cell Biol. 2002;158:647–57.
73. Shi T, Wang F, Stieren E, Tong Q. SIRT3, a mitochondrial
sirtuin deacetylase, regulates mitochondrial function and
thermogenesis in brown adipocytes. J Biol Chem.
74. Rose G, Dato S, Altomare K, Bellizzi D, Garasto S, Greco V,
et al. Variability of the SIRT3 gene, human silent information
regulator Sir2 homologue, and survivorship in the elderly.
Exp Gerontol. 2003;38:1065–70.
75. Bellizzi D, Dato S, Cavalcante P, Covello G, Di Cianni F,
Passarino G, et al. Characterization of a bidirectional
promoter shared between two human genes related to aging:
SIRT3 and PSMD13. Genomics. 2007;89:143–50.
76. Haigis MC, Mostoslavsky R, Haigis KM, Fahie K,
Christodoulou DC, Murphy AJ, et al. SIRT4 inhibits
glutamate dehydrogenase and opposes the effects of calorie
restriction in pancreatic beta cells. Cell. 2006;126:941–54.
77. Mostoslavsky R, Chua KF, Lombard DB, Pang WW,
Fischer MR, Gellon L, et al. Genomic instability and aging-
like phenotype in the absence of mammalian SIRT6. Cell.
78. Liszt G, Ford E, Kurtev M, Guarente L. Mouse Sir2
homolog SIRT6 is a nuclear ADP-ribosyltransferase. J Biol
Chem. 2005;280:21313–20.
79. Grummt I, Pikaard CS. Epigenetic silencing of RNA
polymerase I transcription. Nat Rev Mol Cell Biol. 2003;4:
80. Ford E, Voit R, Liszt G, Magin C, Grummt I, Guarente L.
Mammalian Sir2 homolog SIRT7 is an activator of RNA
polymerase I transcription. Genes Dev. 2006;20:1075–80.
81. Muth V, Nadaud S, Grummt I, Voit R. Acetylation of
TAF(I)68, a subunit of TIF-IB/SL1, activates RNA poly-
merase I transcription. EMBO J. 2001;20:1353–62.
Sirtuins and metabolism 345
Ann Med Downloaded from by Universite Louis Pasteur on 06/17/10
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... Companies were formed, activators were sought, mouse models were created, review articles were written, grants were funded, and laboratories globally were mobilized to better understand human SIRT1, which was described as the SIR2 ortholog among a magnificent set of seven sirtuins that would revolutionize human medicine 41 . The amount of global hype around a yeast gene conserved as a family of dominantly acting animal longevity genes cannot be overstated. ...
Full-text available
Hundreds of colleagues, trainees and members of the general public have asked me to evaluate the thesis that sirtuins--genes related to yeast SIR2--are conserved longevity genes. In this review article, I evaluate the data, and make the case that it is straightforward to reject the thesis that sirtuins are longevity genes. The preprint is in peer review. I encourage scientists to read and discuss this review and to provide comments that will help move the field forward.
... Although these enzymes share a catalytic core domain and a NAD+ binding domain, they also present some differences in their sequence that are responsible for their intracellular function and localization, as well as for their target specificity [187]. Sirtuins were first characterized as histone deacetylases (HDACs), but subsequent and more detailed studies have underlined the presence of non-histonic targets [188]. According to their cellular localization, sirtuins are classified as nuclear (SIRT1, SIRT6), nucleolar (SIRT7), mitochondrial (SIRT3, SIRT4, SIRT5), and cytoplasmatic (SIRT2) [189]. ...
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Epithelial–mesenchymal transition (EMT), a physiological process during embryogenesis, can become pathological in the presence of different driving forces. Reduced oxygen tension or hypoxia is one of these forces, triggering a large number of molecular pathways with aberrant EMT induction, resulting in cancer and fibrosis onset. Both hypoxia-induced factors, HIF-1α and HIF-2α, act as master transcription factors implicated in EMT. On the other hand, hypoxia-dependent HIF-independent EMT has also been described. Recently, a new class of seven proteins with deacylase activity, called sirtuins, have been implicated in the control of both hypoxia responses, HIF-1α and HIF-2α activation, as well as EMT induction. Intriguingly, different sirtuins have different effects on hypoxia and EMT, acting as either activators or inhibitors, depending on the tissue and cell type. Interestingly, sirtuins and HIF can be activated or inhibited with natural or synthetic molecules. Moreover, recent studies have shown that these natural or synthetic molecules can be better conveyed using nanoparticles, representing a valid strategy for EMT modulation. The following review, by detailing the aspects listed above, summarizes the interplay between hypoxia, sirtuins, and EMT, as well as the possible strategies to modulate them by using a nanoparticle-based approach.
... In case of yeast, overexpression of SIRT2 gene increases its lifespan up to 30% [59]. SIRTs are usually insensitive to TSA [60]. SIRT1 decreases fat accumulation in adipose tissue [61]. ...
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4-Hexylresorcinol (4HR) is amphiphilic organic chemical and auto-regulator for micro-organism. As 4HR administration induces the stress on the endoplasmic reticulum, 4HR changes protein folding. The application of 4HR inhibits NF-κB signal pathway and TNF-α production. In addition, 4HR administration increases VEGF, TGF-β1, and calcification associated proteins. As a consequence, 4HR administration increases angiogenesis and bone formation in wounded area. Strong anti-inflammatory reaction and capillary regeneration in diabetic model demonstrate that 4HR can be applied on many types of surgical wound.
... From the strong effects on the expression of sirtuins in muscle, we may assume adaptive responses on the level of energy metabolism and protein acetylation. Sirtuins are a group of deacetylases and ADP-ribosylases with multiple effects on the level of DNA, RNA, protein, or metabolites in different cellular compartments [51]. In elderly men, resistance exercise training increased serum levels of Sirt1, 3, and 6 [52]. ...
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The somatotropic axis is required for a number of biological processes, including growth, metabolism, and aging. Due to its central effects on growth and metabolism and with respect to its positive effects on muscle mass, regulation of the GH/IGF-system during endurance exercise is of particular interest. In order to study the control of gene expression and adaptation related to physical performance, we used a non-inbred mouse model, phenotype-selected for high running performance (DUhTP). Gene expression of the GH/IGF-system and related signaling cascades were studied in the pituitary gland and muscle of sedentary males of marathon and unselected control mice. In addition, the effects of three weeks of endurance exercise were assessed in both genetic groups. In pituitary glands from DUhTP mice, reduced expression of Pou1f1 (p = 0.002) was accompanied by non-significant reductions of Gh mRNA (p = 0.066). In addition, mRNA expression of Ghsr and Sstr2 were significantly reduced in the pituitary glands from DUhTP mice (p ≤ 0.05). Central downregulation of Pou1f1 expression was accompanied by reduced serum concentrations of IGF1 and coordinated downregulation of multiple GH/IGF-signaling compounds in muscle (e.g., Ghr, Igf1, Igf1r, Igf2r, Irs1, Irs2, Akt3, Gskb, Pik3ca/b/a2, Pten, Rictor, Rptor, Tsc1, Mtor; p ≤ 0.05). In response to exercise, the expression of Igfbp3, Igfbp 4, and Igfbp 6 and Stc2 mRNA was increased in the muscle of DUhTP mice (p ≤ 0.05). Training-induced specific activation of AKT, S6K, and p38 MAPK was found in muscles from control mice but not in DUhTP mice (p ≤ 0.05), indicating a lack of mTORC1 and mTORC2 activation in marathon mice in response to physical exercise. While hormone-dependent mTORC1 and mTORC2 pathways in marathon mice were repressed, robust increases of Ragulator complex compounds (p ≤ 0.001) and elevated sirtuin 2 to 6 mRNA expression were observed in the DUhTP marathon mouse model (p ≤ 0.05). Activation of AMPK was not observed under the experimental conditions of the present study. Our results describe coordinated downregulation of the somatotropic pathway in long-term selected marathon mice (DUhTP), possibly via the pituitary gland and muscle interaction. Our results, for the first time, demonstrate that GH/IGF effects are repressed in a context of superior running performance in mice.
Sirt2 regulates various biological processes by deacetylating target genes. Despite roles in regulating proliferation, cell cycle, and glucose metabolism, which are closely associated with skeletal muscle physiology, Sirt2 functions in this tissue remain unclear. In this study, genetic deletion of Sirt2 delayed muscle regeneration after Notexin-induced muscle injury. Gene expressions of myogenic regulatory factors, including Myf5, MyoD, and Myogenin, and cell cycle regulators, such as cyclin D1 and CDK2, were repressed in Sirt2 knockout mice after injury. Also, Sirt2 knockout mice presented muscle atrophy after muscle injury which is associated with the down-regulation of anabolic signaling and the up-regulation of catabolic signaling, in particular, increased atrogin1 transcriptional expression. Thus, Sirt2 positively regulated skeletal muscle regeneration after muscle injury by regulating transcriptional expression involved in myogenesis, cell cycle, and anabolic and catabolic signaling. Based on the in vivo analyses, Sirt2 could function as an interventional therapeutic for chronic myopathy, which is characterized by impaired muscle regeneration and muscle atrophy.
Aging is a collection of changes that contribute to decline in maximum function and ultimately death of an organism. This process is controlled and initiated by several mechanisms including telomere shortening, oxidative stress, AMP-activated protein kinase and sirt-1. Several therapies have been reported to relieve the process of aging. Among these, diet therapy seems to be the most appropriate approach. Fruits are an important part of regular diet. They contain several compounds which have potential to handle the problem of aging and its related disorders. The present paper provides a comprehensive review on different factors present in various fruits related to the process of aging together with their antiaging mechanisms.
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Die Fähigkeit sich an die Rotation der Erde und den daraus resultierenden Tag- und Nacht-Rhythmus anzupassen, basiert auf einer komplexen Regulation verschiedener physiologischer Prozesse. Auf molekularer Ebene liegt diesen Prozessen eine Orchestration von Uhr-Genen zugrunde – auch als innere Uhr bezeichnet – die einen aktivierenden bzw. reprimierenden Einfluss auf die Expression einer Vielzahl weiterer Gene hat. Ausgehend von dieser Regulation lassen sich auf unterschiedlichsten Ebenen tageszeitabhängige, wiederkehrende Rhythmen beobachten. Während diese wiederkehrenden Rhythmen auf einigen Ebenen bereits gut erforscht und beschrieben sind, gibt es weitere Ebenen wie den Metabolismus, über die das Wissen bisher noch begrenzt ist. So handelt es sich bei Drosophila beispielsweise um den Organismus, dessen innere Uhr auf molekularer Ebene wahrscheinlich mit am besten charakterisiert ist. Dennoch ist bisher nur wenig über Stoffklassen bekannt, deren Metabolismus durch die innere Uhr kontrolliert wird. Zwar konnte bereits gezeigt werden, dass sich eine gestörte innere Uhr auf die Anlage der Energiespeicher auswirkt, inwiefern dies allerdings einen Einfluss auf dem intermediären Stoffwechsel hat, blieb bisher weitgehend unerforscht. Auch die Frage, welche Metaboliten wiederkehrende, tageszeitabhängige Rhythmen aufweisen, wurde bisher nur für eine begrenzte Anzahl Metaboliten untersucht. Bei der hier durchgeführten Arbeit wurden deshalb zunächst die globalen Metabolit-Profile von Fliegen mit einer auf molekularer Ebene gestörten inneren Uhr (per01) mit Fliegen, die über eine funktionale Uhr verfügen (CantonS), zu zwei Zeitpunkten verglichen. Um die Anzahl der zeitgleich untersuchten Gewebe und somit die Komplexität der Probe zu reduzieren, wurden hierfür die Köpfe von den Körpern der Fliegen getrennt und separat analysiert. Beide Körperteile wurden sowohl auf kleine hydrophile als auch auf hydrophobe Metaboliten hin mittels UPLC-ESI-qTOF-MS untersucht. Die anschließend durchgeführte, statistische Analyse brachte hervor, dass sich Unterschiede zwischen den beiden Fliegenlinien besonders in den Spiegeln der essentiellen Aminosäuren, den Kynureninen, den Pterinaten sowie den Spiegeln der Glycero(phospho)lipiden und Fettsäureester zeigten. Bei den Lipiden zeigte sich, dass die Auswirkungen weniger ausgeprägt für die Anlage der Speicher- und Strukturlipide als für die Intermediate des Lipidabbaus, die Diacylglycerole (DAGs) sowie die Acylcarnitine (ACs), waren. Um zu bestätigen, dass die inneren Uhr tatsächlich einen regulatorischen Einfluss auf die ausgemachten Stoffwechselwege hat, wurden anschließend die Spiegel aller Mitglieder darauf hin untersucht, ob diese wiederkehrende, tageszeitabhängige Schwankungen aufweisen. Hierfür wurden Proben alle zwei Stunden über drei aufeinanderfolgende Tage genommen und analysiert, bevor mittels JTK_CYCLE eine statistische Analyse der Daten durchgeführt und die Metaboliten herausgefiltert wurden, die ein rhythmisches Verhalten bei einer Periodenlänge von 24h zeigten. Hierbei bestätigte sich, dass besonders die Mitglieder des intermediären Lipidmetablismus hiervon betroffen waren. So konnten zwar auch für einige Aminosäuren robuste Rhythmen ausgemacht werden, besonders ausgeprägt waren diese jedoch erneut bei den DAGs und den ACs. Die abschließende Untersuchung letzterer unter Freilaufbedingungen (DD) sowie in per01 brachte hervor, dass die ausgemachten Rhythmen unter diesen Bedingungen entweder nicht mehr detektiert werden konnten oder deutlich abgeschwächt vorlagen. Lediglich zwei kurzkettige ACs zeigten auch unter DD-Bedingungen statistisch signifikante Rhythmen in ihren Spiegeln. Dies spricht dafür, dass neben der Regulation durch die innere Uhr weitere Faktoren, wie beispielsweise das Licht, eine entscheidende Rolle zu spielen scheinen.
Obesity drives an excessive triglycerides accumulation in adipose tissue, which incites immune cell infiltration, causing fibrosis and inflammation, causing local hypoxia in adipocytes, and ultimately insulin resistance. The extracellular matrix (ECM) complex network of proteins and proteoglycans that offer a scaffold for cells controlling differentiation, migration, repair, survival, and development, and ECM remodeling is required for healthy adipose tissue expansion. To understand the molecular mechanism of this process is a challenge in order to prevent or treat metabolic diseases. This chapter describes the different ECM components and their function related to adipose tissue and their contribution to restore or maintain insulin sensitivity and the whole body metabolism.
Currently, the most widespread global ailment is not COVID-19 or any other such devastating infectious diseases. In fact, obesity has been recognized as a prime risk in the development of cardiometabolic diseases (CMD), neurodegenerative diseases (NDD) and cancer and their morbidity and mortality signature. The pathobiology and therapy of obesity and related diseases are immensely complex at the cellular and molecular levels. This scenario raises the question of how such a complexity may be grappled in a more tangible manner. Since 2003, we have been thinking “what nobody has yet thought about that which everybody sees”, namely, metabotrophic factors (MTF or metabotrophins, metabokines). They include mainly (i) the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and (ii) the adipomyokines adiponectin, irisin, BDNF, fibroblast growth factor-21 alike as adipose- and skeletal muscle-derived signaling proteins. Herein, we argue that obesity and related CMD and NDD, particularly Alzheimer’s disease, may be viewed as MTF-deficient diseases. Further studies on MTF signatures and ramifications in these diseases are required. This may open up an intriguing line of scientific enquiry that will ally adipobiologists with neurobiologists and myobiologists in the fight against obesity. These would provide greater insights on how we can make MTF work for the improvement of physiological and psychological quality of human life.
The circadian clock is an essential timekeeper that controls, for humans, the daily rhythm of biochemical, physiological, and behavioral functions. Irregular performance or disruption in circadian rhythms results in various diseases, including cancer. As a factor in cancer development, perturbations in circadian rhythms can affect circadian homeostasis in energy balance, lead to alterations in the cell cycle, and cause dysregulation of chromatin remodeling. However, knowledge gaps remain in our understanding of the relationship between the circadian clock and cancer. Therefore, a mechanistic understanding by which circadian disruption enhances cancer risk is needed. This review article outlines the importance of the circadian clock in tumorigenesis and summarizes underlying mechanisms in the clock and its carcinogenic mechanisms, highlighting advances in chronotherapy for cancer treatment.
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Studies in invertebrates have led to the identification of a number of genes that regulate lifespan, some of which encode components of the insulin or insulin-like signalling pathways. Examples include the related tyrosine kinase receptors InR (Drosophila melanogaster) and DAF-2 (Caenorhabditis elegans) that are homologues of the mammalian insulin-like growth factor type 1 receptor (IGF-1R). To investigate whether IGF-1R also controls longevity in mammals, we inactivated the IGF-1R gene in mice (Igf1r). Here, using heterozygous knockout mice because null mutants are not viable, we report that Igf1r(+/-) mice live on average 26% longer than their wild-type littermates (P < 0.02). Female Igf1r(+/-) mice live 33% longer than wild-type females (P < 0.001), whereas the equivalent male mice show an increase in lifespan of 16%, which is not statistically significant. Long-lived Igf1r(+/-) mice do not develop dwarfism, their energy metabolism is normal, and their nutrient uptake, physical activity, fertility and reproduction are unaffected. The Igf1r(+/-) mice display greater resistance to oxidative stress, a known determinant of ageing. These results indicate that the IGF-1 receptor may be a central regulator of mammalian lifespan.
Recent studies of obesity show that fat tissue fulfills an endocrine function by producing a variety of secreted proteins, called adipocytokines, that may play key metabolic roles. The present investigators have isolated a newly identified adipocytokine, visfatin, from visceral fat of both mice and humans. Expression of visfatin in the plasma increases as obesity develops. This substance corresponds to a protein identified as preB cell colony-enhancing factor (PBEF), a cytokine expressed in lymphocytes. In a study of 101 human males and females, plasma levels of PBEF correlated closely with the amount of visceral fat as estimated by computed tomography. Correlation with the amount of subcutaneous fat was weak. Significant elevations of PBEF mRNA were also found in KKAy mice, which serve as a model for obesity-related type 2 diabetes. These mice become obese at age 6 to 12 weeks and, at the same time, plasma PBEF levels increase significantly, as do levels of PBEF mRNA in visceral fat. Levels in subcutaneous fat change very little. Mice fed a high-fat diet had higher plasma PBEF concentrations than those fed normal chow. When recombinant visfatin was administered intravenously to c57BL/6J mice, plasma glucose decreased within 30 minutes in a dose-dependent manner. The same effect was noted in insulin-resistant obese KKAy mice, mimicking the effect of insulin injection. Visfatin also had insulin-like effects on cultured cells. In both strains of mice, chronic exposure to visfatin, using adenovirus, significantly lowered plasma levels of both glucose and insulin. Visfatin was found to bind to—and activate—the insulin receptor but in a way different from insulin. These studies indicate that visfatin shares properties of insulin both in vitro and in vivo. In addition to helping to understand glucose and lipid homeostasis and adipocyte proliferation, visfatin may prove to be a useful target when developing drug treatments for diabetes.
In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.