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What are the roles of http://en.wikipedia.org/wiki/Sirtuin
in the events of
http://en.wikipedia.org/wiki/Aging and
http://en.wikipedia.org/wiki/Caloric_restriction that may
assist us in
understanding the events that may occur in each event?

Haigis MC, Guarente LP.
Mammalian sirtuins--emerging roles in physiology, aging, and
calorie
restriction.
Genes Dev. 2006 Nov 1;20(21):2913-21.
PMID: 17079682

Sir2 is an NAD-dependent deacetylase that connects
metabolism with longevity
in yeast, worms and flies. Mammals contain seven homologs of
yeast Sir2,
SIRT1-7. Here, we review recent findings demonstrating the
role of these
mammalian sirtuins as regulators of physiology, calorie
restriction, and
aging. The current findings sharpen our understanding of
sirtuins as
potential pharmacological targets to treat the major
diseases of aging.

Silent information regulator (Sir) proteins regulate
lifespan in multiple
model organisms. In yeast, an extra copy of the SIR2 gene
extends
replicative lifespan by 50%, while deleting Sir2 shortens
lifespan
(Kaeberlein et al. 1999). Sir2 silences chromatin, enables
DNA repair, and
is involved in chromosome fidelity during meiosis (Blander
and Guarente
2004). Sir2 promotes longevity by suppressing the formation
of toxic
extrachromosomal rDNA circles (ERCs) in yeast (Sinclair and
Guarente 1997).
The Caenorhabditis elegans ortholog sir-2.1 also extends
worm lifespan
(Tissenbaum and Guarente 2001), but by a distinct mechanism.
Sir-2.1
requires the worm forkhead protein DAF-16 for lifespan
extension (Tissenbaum
and Guarente 2001). While earlier models suggested sir-2.1
might function by
down-regulating insulin signaling, more recent findings show
that sir-2.1
binds to DAF-16, activating it directly (Berdichevsky et al.
2006).
Moreover, sir-2.1 does not respond to changes in insulin
signaling, but,
rather, is activated by stress treatments, such as heat
shock and oxidative
damage (Berdichevsky et al. 2006). Likewise, an increase in
the dosage of
Drosophila Sir2 extends lifespan (Rogina and Helfand 2004).

Although a link between Sir2 and longevity was clear, its
enzymatic activity
remained elusive for years. An early clue came from the
observation that
CobB, an Escherichia coli homolog of Sir2, could catalyze the
phosphoribosyltransferase reaction in cobalamin biosynthesis
(Tsang and
Escalante-Semerena 1998). Thus, it was predicted and
demonstrated that Sir2
possessed NAD-dependent ADP-ribosyltransferase activity
(Frye 1999; Tanny et
al. 1999). Subsequent reports revealed that yeast and
mammalian sirtuins
catalyze a novel and robust reaction, NAD-dependent histone
deacetylation,
unavoidably linking Sir2 activity with metabolism (Imai et
al. 2000; Landry
et al. 2000; Smith et al. 2000). Mechanistically,
ADP-ribosylation and
deacetylation reactions by sirtuins are similar because they
cleave NAD in
each reaction cycle (Fig. 1) (Grubisha et al. 2005). During
each cycle,
deacetylation generates the novel metabolites, 2' and
3'-O-acetyl-ADP-ribose
(Tanner et al. 2000; Sauve et al. 2001; Tanny and Moazed
2001), which may be
important regulators of physiology (Grubisha et al. 2006).
Today, more than
a dozen nonhistone deacetylation substrates are known,
several of which are
described below.

Figure 1. Sirtuin deacetylation and ADP-ribosylation
reactions. Both
deacetylation and ADP-ribosylation occur via cleavage of NAD
to release
nicotinamide.

Calorie restriction (CR) is a dietary regimen that extends
the lifespan of
every organism tested to date. Specifically, CR extends the
lifespan of
yeast (Lin et al. 2002), spiders (Austad 1989), flies (Loeb
and Northrop
1917), fish (Comfort 1963), and rodents (McCay et al. 1935;
Austad 1989).
Sir2 is required for lifespan extension by CR in yeast,
worms, and flies
(Lin et al. 2000; Rogina and Helfand 2004; Wang and
Tissenbaum 2006). In
yeast, CR (0.5% glucose), was previously shown to increase
mitochondrial
function and to up-regulate SIR2 activity (Lin et al. 2002,
2004). However,
in this case, the mitochondrial activation is
SIR2-independent, suggesting
that it lies upstream of SIR2. A more severe CR regimen
(0.05% glucose)
extends yeast replicative lifespan by a different mechanism
that is
apparently independent of both SIR2 and mitochondrial
respiration
(Kaeberlein et al. 2004, 2005). Finally, SIR2 has no effect
on yeast
survival under starvation conditions, and appears to
actually reduce
survival of certain exceptionally long-lived mutant strains
(Fabrizio et al.
2005).

Do sirtuins regulate human longevity? Mammals have seven
Sir2 homologs
(sirtuins, SIRT1-7). These proteins have a highly conserved
NAD-dependent
sirtuin core domain, first identified in the founding yeast
SIR2 protein,
making them good candidates as lifespan regulators (Frye
2000). As
highlighted in this review, mammalian sirtuins have diverse
cellular
locations, target multiple substrates, and affect a broad
range of cellular
functions (Table 1). In this review, we emphasize an
emerging theme in the
field of aging-the regulation of oxidative stress, DNA
damage, and
metabolism by mammalian sirtuins.

Table 1. Diversity of mammalian sirtuins
=====================================================
Sirtuin Activity Location Interactions Biology
SIRT1 Deacetylase Nucleus FOXO, PGC-1alpha Cell
survival/metabolism
SIRT2 Deacetylase Cytosol Tubulin, H4 Cell cycle
SIRT3 Deacetylase Mitochondria AceCS2 Thermogenesis/metabolism
SIRT4 ADP-ribosyl-transferase Mitochondria GDH Insulin
secretion/metabolism
SIRT5 Deacetylase Mitochondria ? ?
SIRT6 ADP-ribosyl-transferase Nucleus DNA Polß DNA repair
SIRT7 ? Nucleolus Pol I rDNA transcription

Nuclear sirtuins

Three mammalian sirtuins (SIRT1, SIRT6, and SIRT7) are
localized to the
nucleus. SIRT1 is most extensively studied, has more than a
dozen known
substrates, and is a guardian against cellular oxidative
stress and DNA
damage. Moreover, SIRT1 plays a prominent role in metabolic
tissues, such as
pancreas, fat, and liver. SIRT6 and SIRT7 may also be
important regulators
of DNA damage and metabolism, respectively.

SIRT1 substrates in mammals

While a role for mammalian sirtuins in lifespan regulation
has not been
directly determined, evidence suggests that the Sir2
ortholog, SIRT1, may
regulate many physiological processes known to be affected
during aging and
which are altered by CR (Fig. 2). SIRT1 deacetylates a large
number of
substrates, including p53, Ku70, NF-B, and forkhead proteins
to affect
stress resistance in cells (Luo et al. 2001; Vaziri et al.
2001; Brunet et
al. 2004; Cohen et al. 2004; Motta et al. 2004; Yeung et al.
2004), which
may relate to the observed stress resistance conferred by
CR. SIRT1 also
regulates the activities of the nuclear receptor PPAR and
PGC- (see below)
to influence differentiation of muscle cells, adipogenesis,
fat storage in
white adipose tissue, and metabolism in the liver,
suggesting a possible
connection between this sirtuin and diets that promote
leanness and
longevity (Fulco et al. 2003; Picard et al. 2004; Rodgers et
al. 2005). The
observed induction of SIRT1 protein during CR is consistent
with this idea
(Cohen et al. 2004; Nisoli et al. 2005). These activities
link SIRT1 to
known physiological effects of CR, and suggest that this
sirtuin may help
mediate CR in mice.

Figure 2. SIRT1 regulation of mammalian physiology.
SIRT1 regulates
neuron survival, gluconeogenesis, lipolysis, ß-cell
survival, and insulin
secretion by interacting with a number of target proteins.

SIRT1 regulation of insulin and glucose homeostasis

A critical component of the physiology of CR is increased
insulin
sensitivity and corresponding reductions in blood glucose
and insulin levels
(Barzilai et al. 1998; Dhahbi et al. 2001). Pancreatic
ß-cells help to
maintain glucose homeostasis by secreting insulin in
response to glucose.
Metabolism of glucose in these cells by glycolysis generates
pyruvate, which
enters mitochondria where it can be converted to CO2 by the
TCA cycle. NADH
made by this metabolic process drives electron transport and
ATP synthesis.
The increased ATP/ADP ratio causes closure of KATP channels
and depolarizes
the plasma membrane leading to an influx of Ca2+, which
triggers fusion of
secretory vesicles containing insulin to the cell membrane.

Two recent studies in mice have demonstrated that SIRT1
positively regulates
glucose-stimulated insulin secretion in pancreatic ß-cells
(Moynihan et al.
2005; Bordone et al. 2006). ß-Cell-specific
SIRT1-overexpressing (BESTO)
mice demonstrate increased insulin secretion in response to
glucose
(Moynihan et al. 2005). Conversely, SIRT1-/- mice or their
isolated islets
show blunted insulin secretion (Bordone et al. 2006). Both
studies find that
SIRT1 represses transcription of the mitochondrial
uncoupling protein UCP-2
gene, which uncouples mitochondrial respiration from ATP
production and
reduces the proton gradient across the mitochondrial
membrane. Thus, by
blocking UCP-2 function, SIRT1 promotes more efficient
energy generation.
Indeed, BESTO islets demonstrate higher ATP levels (Moynihan
et al. 2005),
while islets from SIRT1 knockout (KO) mice do not elevate
ATP production in
response to glucose (Bordone et al. 2006). Interestingly,
SIRT1-mediated
repression of UCP-2 is alleviated by acute food deprivation
(Bordone et al.
2006), which may further dampen ATP synthesis and the
insulin responsiveness
of ß-cells during starvation. While SIRT1 protein level is
not affected by
this condition, there is a decrease in the NAD/NADH ratio,
which may reduce
SIRT1 activity in pancreas (Bordone et al. 2006). The
presence of UCP-2 in
fasted animals may also ease the transition to metabolic
activity after the
next feeding and prevent hyperpolarization of the
mitochondrial membrane and
the corresponding production of reactive oxygen species.
While these studies
demonstrate that SIRT1 activity may be down-regulated in
ß-cells during
fasting, it is not known whether SIRT1 regulates insulin
secretion during
CR, or plays any role in pathologies demonstrating impaired
insulin
secretion.

Another study raises the possibility that SIRT1 promotes the
survival of
pancreatic ß-cells during oxidative stress (Kitamura et al.
2005). In
stressed ß-cells, the forkhead protein FOXO1 moves into the
nucleus and
activates the ß-cell transcription factors, NeuroD and MafA,
and provides
stress resistance. As described above, SIRT1 binds to and
regulates forkhead
transcription factors both negatively and positively (Brunet
et al. 2004;
Motta et al. 2004). Kitamura et al. (2005) show that nuclear
FOXO1
associates with SIRT1 in PML (promyelocytic leukemia) bodies
in stressed
cells. They suggest that SIRT1 deacetylates FOXO1 at that
location to
activate the protein and provide stress resistance (Accili
and Arden 2004).
This process bears similarity to sir-2.1 in C. elegans
discussed above,
which functions as a coactivator of the DAF-16 protein after
it translocates
to the nucleus in oxidatively or heat stressed worms
(Berdichevsky et al.
2006).

Glucose homeostasis is maintained by the liver, in addition
to pancreatic
ß-cells, in response to changing nutrient conditions. During
fasting,
hepatocytes induce gluconeogenesis to supply other tissues
with glucose.
Several new studies have revealed that this nutrient
response is under tight
control of SIRT1 activity, providing another link between
SIRT1 and
metabolism. In cultured hepatocytes, SIRT1 interacts with
and deacetylates
nuclear FOXO1, promoting FOXO1-dependent transcription of
hepatic
gluconeogenic genes upon stress (Frescas et al. 2005). In
the liver, the
transcriptional coactivator PGC-1 also drives expression of the
gluconeogenic pathway. SIRT1 deacetylates and activates
PGC-1 to coordinate
the increase in expression of gluconeogenic genes with the
repression of
glycolytic genes during fasting (Rodgers et al. 2005).
However, in a
neuronal cell line, overexpression of SIRT1 decreases the
activity of PGC1-
and mitochondrial function (Nemoto et al. 2005), suggesting
that the
relationship between SIRT1 and PGC-1 may be complex.

SIRT1 and neuron function

SIRT1 has also been linked to the survival of neurons. It is
interesting to
note that CR protects against neurodegenerative pathology in
mouse models
for Alzheimer's (Zhu et al. 1999; Patel et al. 2005) and
Parkinson's (Duan
and Mattson 1999). SIRT1 can promote survival in cultured
neuronal cells as
an antiapoptotic factor, perhaps through down-regulating the
proapoptotic
factors, p53 (Luo et al. 2001; Vaziri et al. 2001) and FOXO
(Brunet et al.
2004; Motta et al. 2004). Even more interestingly, SIRT1 may
be involved in
the axonal protection observed in the Wallerian strain of
mice (Araki et al.
2004), which have a translocation that increases levels of
the NAD
biosynthetic enzyme nicotinamide mononucleotide
adenylyl-transferase 1 and
renders peripheral axons more stable after a neuronal
insult. Indeed, NAD
itself provides protection to axons in cultured dorsal root
ganglia. One
study shows that the effects of NAD and the Wallerian strain
are dependent
on SIRT1, leading to the conclusion that this sirtuin is
neuroprotective
(Araki et al. 2004). However, another study did not observe
a difference in
the response of dorsal root ganglion from SIRT1 KO mice
(Wang et al. 2005).
It is interesting to note that the time course of axonal
degradation in the
two studies is different, suggesting that there may be two
different
neuroprotective processes induced by NAD- one dependent on
SIRT1 and one
not. Future studies should resolve these apparent experimental
discrepancies.

SIR2 orthologs can also protect against neuronal dysfunction
due to
polyglutamine toxicity in C. elegans and mammalian cells
(Parker et al.
2005). One study shows that neurotoxicity in worms is spared
by the age-1
mutation, which reduces insulin-like signaling (Morley et
al. 2002), or in a
transgenic strain overexpressing sir-2.1 (Parker et al.
2005). Like the
effects of the age-1 or sir-2.1 transgenes in extending
lifespan,
neuroprotection requires the forkhead protein DAF-16 (Parker
et al. 2005).
As might be expected, polyglutamine toxicity is exacerbated
in daf-16 or
sir-2.1 mutants. The putative SIR2-activating polyphenol,
resveratrol
(Howitz et al. 2003), also protects against cell death in
striatal neurons
with the Huntingtons Disease allele htt (109Q) (Parker et
al. 2005).
Finally, ß-amyloid-induced death of microglia is spared by
overexpression of
SIRT1 or resveratrol treatment (J.Chen et al. 2005). In
toto, the above
studies raise the possibility that activation of SIRT1 may
be a novel
strategy to protect against neurodegenerative diseases. We
note, however,
that the protective effect of SIRT1 may not be universal in
all cell types,
since SIRT1-/- mouse embryonic fibroblasts (MEFs) actually
survive better in
culture and bypass senescence (Chua et al. 2005).

SIRT1 and CR

The above studies suggest that important physiological
processes triggered
by CR in mammals are regulated by SIRT1, making it vital to
know whether
mammalian sirtuins also regulate changes during CR. Recent
experiments have
directly related SIRT1 function to CR in mice. CR induces
the endothelial
nitric oxide synthase (eNOS), and results in an increase in
mitochondrial
biogenesis (Nisoli et al. 2005). Moreover, this
mitochondrial induction by
CR does not occur in eNOS deficient mice. Interestingly, the
SIRT1 gene is
activated by NO in vivo and in vitro (Nisoli et al. 2005),
tracing a pathway
in which CR induces NO production and activates
mitochondrial biogenesis and
SIRT1.

A more direct demonstration of the requirement of SIRT1 in
CR involved
placing SIRT1 KO mice on this diet (D.Chen et al. 2005).
Although the KO
mice show changes in blood glucose, triglycerides, and IGF-1
similar to
wild-type controls, there is a large difference in one
interesting output of
CR. Wild-type mice show a fivefold to 10-fold increase in
physical activity,
which has been observed previously and may represent a
foraging instinct
induced by food insufficiency, but KO mice do not display
any increase in
activity. SIRT1 KO mice move as well or better than wild
type when
challenged by other means; i.e., rotarod or treadmill. This
study indicates
the first requirement for SIRT1 for at least one phenotype
triggered by
mammalian CR.

SIRT6 regulates DNA repair

SIRT6 is a nuclear protein widely expressed in mouse tissues
(Liszt et al.
2005; Michishita et al. 2005). Original reports demonstrated
that SIRT6 has
a weak to absent in vitro deacetylate activity (North et al.
2003; Liszt et
al. 2005). However, SIRT6 has also been shown to demonstrate
a robust
auto-ADP-ribosyltransferase activity (Liszt et al. 2005).

Recent work has provided insight into the diverse
physiological functions of
SIRT6 (Mostoslavsky et al. 2006). SIRT6 KO mice display
premature aging
symptoms, including loss of subcutaneous fat and decreased
bone density, and
die within 4 wk after birth. These phenotypes contrast with
those of SIRT1
KO animals, which are postnatal lethal on an inbred strain
(Cheng et al.
2003; McBurney et al. 2003). Outbred SIRT1 KO animals can
survive into
adulthood, but demonstrate a severe phenotype, including
small size, delayed
bone mineralization, defective skeletal closure, delayed
eyelid opening, and
sterility (Cheng et al. 2003; McBurney et al. 2003; Lemieux
et al. 2005).

SIRT6 KO mice exhibit a deficiency in one specific form of
DNA repair, the
base excision repair (BER) (Mostoslavsky et al. 2006). MEFs
lacking SIRT6
demonstrate impaired proliferation and enhanced sensitivity
to DNA-damaging
agents. SIRT6 KO MEFs demonstrate genomic instability in the
form of
chromosomal translocations, fragments, gaps, and detached
centromeres. These
defects can be rescued by overexpression of the DNA
polymerase involved in
BER, Polß. Furthermore, SIRT6 KO MEFs exhibit normal cell
cycle checkpoints,
end-joining, and double-strand break DNA repair. How SIRT6
regulates BER is
still unknown. One might hypothesize that this sirtuin
ADP-ribosylates a
substrate protein involved in BER, which could be a
component of the repair
machinery or the chromatin at the site of DNA damage.

SIRT6 KO mice also display interesting metabolic phenotypes:
low levels of
circulating IGF-1 and hypoglycemia that becomes
progressively more severe
with age (Mostoslavsky et al. 2006). It will be interesting
to know whether
these metabolic changes are due to a direct role for SIRT6
in regulating
IGF-1 and glucose homeostasis or are an indirect consequence
of DNA damage
that accumulates in these mutant mice.

SIRT7 promotes rRNA transcription

SIRT7 localizes to the nucleolus of human cells (Michishita
et al. 2005;
Ford et al. 2006). Interestingly, SIRT7 expression
correlates with growth
(Ford et al. 2006)-it is abundant in tissues with high
proliferation, such
as liver, spleen, and testes. By contrast, SIRT7 expression
is absent or low
in nonproliferating tissues, like heart, brain, and muscle.

Recent work has shown that SIRT7 may regulate cellular
growth and metabolism
(Ford et al. 2006). In the nucleolus, SIRT7 associates with
rDNA and
interacts with RNA polymerase I (Pol I). Overexpressing
SIRT7 increases rRNA
transcription and RNA inhibition of SIRT7 decreases
transcription, showing
that this sirtuin activates Pol I transcription (Ford et al.
2006).

An NAD-dependent deacetylase activity has not been observed
for SIRT7 (North
et al. 2003), but the amino acid residues that bind NAD in
the conserved
sirtuin core domain are required for SIRT7 activity (Ford et
al. 2006),
suggesting a role for NAD-dependent regulation. SIRT7 thus
appears to
regulate cell growth and metabolism in response to changing
metabolic
conditions by driving ribosome biogenesis in dividing cells.
It is
interesting that both SIRT7, as an activator of rRNA
transcription, and
SIRT1, as an inhibitor of p53 and FOXO, have features that
are progrowth and
prosurvival for cells.

Cytoplasmic sirtuins

To date, only SIRT2 is reported to be localized mainly in
the cytoplasm
(North et al. 2003; Michishita et al. 2005), while a
fraction of SIRT2 is
nuclear (North et al. 2003). Interestingly, SIRT1 is also
reported to be a
cytoplasmic protein in pancreatic -cells (Imai et al. 2000).
These findings
lead us to speculate that mammalian sirtuins may shuttle
between the nucleus
and cytoplasm, depending on cell type or environmental stimuli.

SIRT2

Mammalian SIRT2 is a predominantly cytoplasmic protein
(Dryden et al. 2003;
North et al. 2003; Michishita et al. 2005), colocalizes with
tubulin, and
can deacetylate a number of substrates in vitro, including
a-tubulin (North
et al. 2003) and histones, although the physiological
consequences of
a-tubulin deacetylation by SIRT2 are not yet clear. The
yeast ortholog of
SIRT2, Hst2, can function in parallel to SIR2 in certain
strains with
respect to lifespan extension and rDNA silencing (Lamming et
al. 2005).
Therefore, it will be interesting to determine the lifespan of
SIRT2-overexpressing mice, or to determine whether CR is
partly mediated by
SIRT2 using SIRT2 KO animals.

Cell culture studies demonstrate SIRT2 may be important in
regulating
mammalian cell cycle. SIRT2 protein levels increase during
mitotic phase of
the cell cycle and its overexpression delays mitosis (Dryden
et al. 2003).
Consistent with the idea that SIRT2 may restrain the cell
cycle, expression
of this sirtuin is down-regulated in human gliomas, compared
with normal
brain samples (Hiratsuka et al. 2003). SIRT2 colocalizes
with chromatin
during the G2/M transition, a period in which the nuclear
membrane has
broken down (Vaquero et al. 2006). Both SIRT2 and Hst2 show
a preference for
deacetylating histone H4 at Lys16 in vitro, and SIRT2 KO
mouse embryonic
fibroblasts (MEFs) display hyperacetylated H4K16 during
mitosis. Since SIRT1
also deacetylates H4K16, SIRT2 and SIRT1 may function
redundantly, at least
during the M phase of the cell cycle. SIRT2 may also
regulate other phases
of the cell cycle, since G1 is extended and S is shortened
in SIRT2-/-MEFs.

Mitochondrial sirtuins-key regulators of metabolism

Mitochondria are dynamic organelles that regulate nutrient
utilization to
provide the cell with energy even during dramatic changes in
diet and
development. Mitochondria also play a central role in
mediating apoptosis in
response to DNA damage or oxidative stress. These organelles
are the primary
site of reactive oxygen species (ROS) generation within the
cell, and
increased oxidative damage is proposed to be one cause of
mammalian aging
(Harmon 1956; Wallace 2005).

The mitochondrial localization of SIRT3-5 is especially
intriguing because
mitochondrial dysfunction is associated with mammalian aging
and many
diseases, including diabetes, neurodegenerative diseases,
and cancer
(Wallace 2005). Do mitochondrial sirtuins regulate
metabolism, the oxidative
stress response, and ultimately, mammalian aging? It is
important to note
that although SIRT1 is not itself physically associated with
mitochondria,
as described above, it also impacts mitochondrial functions.
Lifespan
analysis of animals with varying SIRT3-5 level has not been
performed;
however, there is growing evidence linking mitochondrial
sirtuins with
regulating energy usage and even human lifespan.

SIRT3

SIRT3 was the first sirtuin shown to be localized to the
mitochondria of
mammalian cells (Onyango et al. 2002; Schwer et al. 2002;
Michishita et al.
2005). SIRT3 is localized to the mitochondrial matrix and
cleavage of its
signal sequence is necessary for enzymatic activity (Schwer
et al. 2002).
SIRT3 deacetylates multiple substrates in vitro including
histone peptides
(Onyango et al. 2002; Schwer et al. 2002) and tubulin (North
et al. 2003).

The biological functions of SIRT3 are beginning to emerge.
SIRT3 is
expressed in brown adipose tissue and induced by cold
exposure (Shi et al.
2005). Moreover, the deacetylase activity of SIRT3 is
reported to be
required for the induction of uncoupling protein 1 (UCP-1).
SIRT3 also
appears to regulate mitochondrial functions, as its
overexpression increases
respiration, while decreasing reactive oxygen species
production (Shi et al.
2005).

Two recent studies demonstrate that SIRT3 may regulate the
activity of
acetyl-CoA synthetase (AceCS) (Hallows et al. 2006; Schwer
et al. 2006),
representing a striking, conserved activity with the
bacterial sirtuin, cobB
(Tsang and Escalante-Semerena 1998; Starai et al. 2002).
AceCS uses acetate,
CoA, and ATP to form acetyl-CoA, which is an intermediate in
the TCA cycle,
and is also required for cholesterol and fatty acid
synthesis. Acetylation
of mitochondrial AceCS (AceCS2) inactivates the enzyme, whereas
deacetylation by SIRT3 activates it. Interestingly, SIRT1
can deacetylate
and activate the cytosolic form of AceCS (AceCS1). These
data suggest that
SIRT3 may play a role in regulating the entry of carbons
from acetate into
central metabolism. It will be important to assess the in
vivo relevance of
these findings using SIRT3-/- mice. In sum, SIRT3 may be
especially
important under conditions of energy limitation-i.e., during
fasting or CR
to ensure full incorporation of dietary or ketone-derived
acetate into
metabolism.

In human population studies, polymorphisms within the SIRT3
gene have been
linked to longevity. The G477T transversion, while not
affecting the amino
acid sequence, associates with survivalship of elderly males
(Rose et al.
2003) and may signify a haplotype promoting longevity. The
same group found
that a variable number of tandem repeats (VNTR) enhancer
within SIRT3 also
associates with lifespans >90 yr (Bellizzi et al. 2005).
These findings will
need to be validated in larger samples, but suggest that the
expression of
SIRT3 may promote longevity in humans and raise the
importance of performing
lifespan experiments in mice that overexpress or lack SIRT3.

SIRT4

SIRT4 is another mitochondrial protein (Michishita et al.
2005) that
regulates energy usage. SIRT4 lacks detectable deacetylase
activity (North
et al. 2003), but demonstrates ADP-ribosyltransferase
activity. SIRT4 plays
an important role in regulating amino acid-stimulated
insulin secretion
(AASIS) in pancreatic ß-cells by ADP-ribosylating and
inhibiting glutamate
dehydrogenase (GDH) (Fig. 3; Haigis et al. 2006). GDH
converts glutamate
into -ketoglutarate, a TCA cycle intermediate.
GDH-activating mutations
cause hyperinsulinism in humans showing that this enzyme
regulates insulin
secretion by gating the flow of amino acids into central
metabolism in
ß-cells (Stanley et al. 1998). SIRT4 KO mice have no gross
abnormalities,
but display higher GDH activity and higher levels of
circulating insulin.
SIRT4 KO mice have elevated AASIS, and strikingly, unlike
wild type, they
secrete insulin in response to glutamine.

Figure 3. Model of SIRT4 function in pancreatic ß-cells
and liver. SIRT4
ADP-ribosylates and inhibits GDH. In pancreatic ß-cells,
SIRT4 thus
regulates AASIS. In liver, SIRT4 may also regulate the
metabolism of amino
acids to glucose.

SIRT4 may also connect insulin secretion with CR. Islets
isolated from CR
mice demonstrate increased AASIS similar to islets from ad
libitum fed SIRT4
KO mice (Haigis et al. 2006). Down-regulation of SIRT4
during CR appears to
mediate this effect because GDH from islets of CR mice is less
ADP-ribosylated and more active than GDH from control
islets. A similar
change in GDH is found in the liver of CR mice. We suggest
that SIRT4
coordinates a physiological response in liver and ß-cells
during energy
limitation (Fig. 3). In the liver, the flow of carbon from
amino acids into
gluconeogenesis would be increased, and in ß-cells the
ability amino acids
to trigger insulin secretion would be elevated. Since
overall insulin
secretion is clearly lower during CR because of reduced
blood glucose, it
seems likely that the spectrum of insulin secretagogues is
thus shifted from
carbohydrates to amino acids.

We note that the apparent down-regulation of SIRT4 during CR
in ß-cells and
liver goes against the expectation that sirtuin activity
should increase
during this dietary regimen. However, it is consistent with
the observed
reduction in the NAD/NADH ratio in liver of CR mice
(Hagopian et al. 2003b)
and the observed increase in gluconeogenesis in this organ
(Hagopian et al.
2003a). We suggest that a shift from carbohydrates to fat as
preferred
energy source during CR may help drive down the NAD/ NADH
ratio in these
tissues and thus moderate these metabolic changes.

SIRT5

SIRT5 remains the least characterized sirtuin to date. SIRT5
is described as
a mitochondrial protein (Michishita et al. 2005) and has
weak deacetylase
activity (North et al. 2003), but does not appear to possess an
ADP-ribosyltransferase activity (Haigis et al. 2006).
Possible physiological
substrates of SIRT5 and its role in mammalian cells are not
yet known.

Summary and perspective

Sirtuins have emerged as key antiaging genes in model
organisms. The
NAD-dependence of these proteins links them unavoidably to
the metabolic
activity of cells. In several organisms, sirtuins have been
shown to be
regulated by and to mediate the effects of the dietary
regimen CR. Moreover,
mammalian sirtuins have been implicated in stress resistance
and numerous
metabolic pathways, including adipogenesis, gluconeogenesis,
and insulin and
glucose homeostasis.

While it may be years before we know whether sirtuins
regulate mammalian
lifespan, current data suggests that these proteins are
regulated by diet
and in turn, regulate multiple facets of physiology, making
them interesting
therapeutic targets for metabolic and neurodegenerative
diseases (Fig. 4).
Several important questions need to be resolved to improve
our understanding
of sirtuin biology and their therapeutic potential. First,
what other
functions can be ascribed to the SIRT1-7 proteins? We
predict that like
SIRT1, SIRT2-7 will have multiple targets and may affect
many biologies.
Second, how is sirtuin function regulated? Two known
mechanisms of sirtuin
regulation are (1) its protein induction during CR or
fasting (Cohen et al.
2004; Nisoli et al. 2005; Rodgers et al. 2005) and (2)
regulation by
metabolites NAD, NADH, and nicotinamide (Anderson et al.
2003a, b; Lin et
al. 2004). Third, are all seven sirtuins regulated in the
same direction by
CR in any given tissue? If the NAD/ NADH ratio (or nicotinamide
concentration) is a primary determinant of regulation, it is
possible that
all seven sirtuins will be regulated similarly by CR in a
given cell, since
the NAD/NADH ratio has the potential to equilibrate
throughout cellular
compartments by shuttle systems. Another likely possibility
is that the
concentration of NAD and its metabolites are regulated
compartmentally. For
example, enzymes involved in NAD biosynthesis are found in
the nucleus,
peroxisome, Golgi apparatus, mitochondria, and cytoplasm
(Yang et al. 2006).
Fourth, does the NAD/NADH ratio change in different
directions depending on
the tissue in response to CR? This possibility seems likely,
since CR
induces distinct metabolic changes in different tissues
(e.g., activation of
gluconeogenesis in liver and fat loss in white adipose
tissue). Moreover,
fasting decreases SIRT1 activity in the pancreas (Bordone et
al. 2006),
while increasing its activity in the liver (Rodgers et al.
2005). This last
question is important, because if sirtuins are activated by
CR in some
tissues but repressed in others, genetically altered mice
(i.e., SIRT
knockout or overexpressed) or pharmacological interventions
to activate or
repress a sirtuin systemically (see below) may mimic CR in
only a segmental
fashion.

Figure 4. Model of Sirtuin regulation and function.
Changes in
environmental stress and diet may regulate sirtuin activity
by altering the
NAD/NADH ratio, nicotinamide levels, or sirtuin levels.
Mammalian sirtuins
(SIRT1-7) have individual targets and roles in the nucleus,
cytoplasm, or
mitochondria, and may uniquely impact metabolism, DNA
repair, or cell
survival.

In the next few years the answers to these and other
questions will auger
how well pharmacological agents that target sirtuins will
serve as CR
mimetics. This path of drug intervention is especially
compelling, because
CR mitigates many major diseases in rodent models. We can
hope that new
classes of drugs are on the horizon to deliver broad
benefits for these
diseases.