**3. Sirtuin-dependent metabolic regulation**

As its can be concluded by the complexity of sirtuin interactions, the different partners and regulatory processes, this family of deacetylases is involved in many different physiological mechanisms in cells. In the following sections we are going to resume the most important findings about the role of these enzymes in metabolic control in relationship with the aging process. Taken into consideration that metabolic processes are involved in all the cellular processes, metabolic control by sirtuins is the most important function of these enzymes (Yu & Auwerx, 2009).

#### **3.1 SIRT1**

Looking inside this deacetylase enzymes family, SIRT1 is one of the members that show more interactions and that respond to more factors. Mammalian SIRT1 has multiple targets including histones, transcription factors and other molecules that collectively modulate

Other important issue in the regulation of sirtuin levels is the complex and new world of microRNAs (miR)-dependent regulation. Currently, the study of the regulation of sirtuin expression by microRNAs has demonstrated that these proteins are also regulated by this system. MiR-34a is markedly reduced in p53-null PC3 cells and its overexpression inhibits SIRT1 expression at the transcriptional level indicating a p53-dependent regulation of SIRT1 levels (Fujita et al., 2008). On the other hand, in mesenchimal transition processes in breast cancer, the downregulation of miR-200 has been related to the increase in the levels of SIRT1 in these cells contributing to the tumoral phenotype (Eades et al., 2011). On the other hand, the release of proinflammatory mediators in adipocytes in serum-free conditions is regulated by the inhibition of SIRT1 expression mediated by miR-132 (Strum et al., 2009). Further, miR-199a also represses SIRT1 in cardiomyocytes and its downregulation in low oxygen tension conditions derepresses SIRT1 expression at the same time than HIF-1α (Rane et al., 2009). Other interesting miRs, miR-33a and b, are involved in the regulation of fatty acid oxidation including the levels of SIRT6. Increase in the levels of miR-33a and b decrease fatty acid oxidation and also insulin signaling in hepatic cell lines indicating a regulatory role of these miRs in important metabolic pathways in the cell (Davalos et al., 2011). Taken together, it is clear that the, to date, poorly clarified regulatory mechanisms depending on miRs complicate the regulatory mechanisms of sirtuin levels at posttranscriptional level. During last years, small polyphenol molecules have also demonstrated capacity to increase sirtuin activity. Some years ago, we and others demonstrated that resveratrol, a polyphenol of the family of stilbenes found in grapes, dry fruits and berries, is able to extend lifespan in mice fed under high fat conditions (Baur et al., 2006). In this process, sirtuin activity was considered as an important factor. From them, several other works have demonstrated the importance of resveratrol and related compounds in sirtuin-dependent metabolic modifications. In fact, resveratrol is able to modulate insulin response (Zhang, 2006), and also regulate AMPK activity (Dasgupta & Milbrandt, 2007). In some cases, these effects have been related to sirtuin activity and in others, a sirtuin-independent effect has been suggested. In any case, in our hands resveratrol have shown capacity to increase SIRT1 and SIRT3 levels in cultured cells indicating the capacity to modulate sirtuin expression (Santa-Cruz Calvo et al., unpublished results), accordingly with already published results (Costa

As its can be concluded by the complexity of sirtuin interactions, the different partners and regulatory processes, this family of deacetylases is involved in many different physiological mechanisms in cells. In the following sections we are going to resume the most important findings about the role of these enzymes in metabolic control in relationship with the aging process. Taken into consideration that metabolic processes are involved in all the cellular processes, metabolic control by sirtuins is the most important function of these enzymes (Yu

Looking inside this deacetylase enzymes family, SIRT1 is one of the members that show more interactions and that respond to more factors. Mammalian SIRT1 has multiple targets including histones, transcription factors and other molecules that collectively modulate

Cdos et al., 2011; Kao et al., 2010; Sulaiman et al., 2010).

**3. Sirtuin-dependent metabolic regulation** 

& Auwerx, 2009).

**3.1 SIRT1** 

several processes such as energy metabolism, stress response and cell survival (Tang, 2011). Its activity may decline with aging in many tissues and it has been proposed that its reactivation can produce beneficial effects (Tang, 2011).

One of the most important factors involved in the metabolic control regulated by SIRT1 is PGC1α. SIRT1 functionally interacts with PGC1α and deacetylates it (Nemoto et al., 2005). Deacetylation of PGC1α activates this transcription factor that induce the expression of nuclear respiratory factor 1 (NRF1) and then, mitochondrial biogenesis. In fact, activation of SIRT1 induces deacetylation of PGC1α and FOXO1 that finally control the transcriptional modulation for lipid catabolism (Canto et al., 2010). Further, deletion of SIRT1 alters fatty acid metabolism resulting in hepatic steatosis and inflammation (Purushotham et al., 2009). SIRT1-dependent regulatory mechanisms regulate the switch from carbohydrate to lipid as main energy sources in muscle. Limitation in glucose availability during fasting or exercise induces AMPK activity in muscle that acts as a prime initial sensor that activates SIRT1. PGC1α is acetylated by the acetyltransferase GCN5 that together with SIRT1 control its regulation depending on nutritional status (Dominy et al., 2010). Activity of this GCN5 or inhibition by nicotinamide reduces SIRT1-dependent PGC1α acetylation and decreases the expression of genes involved in mitochondrial biogenesis in muscle (Gerhart-Hines et al., 2007). Further, PGC1β is also acetylated on at least 10 lysine residues by GCN5 repressing its transcriptional activity, SIRT1 activity also deacetylates it and restores transcriptional activity (Kelly et al., 2009).

As a cycle of regulation, SIRT1 also controls the expression levels of PGC1α in skeletal muscle through stimulation of its promoter activity probably with the activity of myogenic factors such as MEF2 and MyoD (Amat et al., 2009). On the other hand, PGC1α is also involved in sirtuin expression since, as mitochondrial biogenesis is activated and some sirtuins are located in mitochondria, the expression of SIRT3 gene is also controlled by PGC1α. This regulation is key in the differentiation of brown adipocytes (Giralt et al., 2011).

Besides the high number of evidences demonstrating the relationship of SIRT1 activity and PGC1α-dependent mitochondrial biogenesis, some other works indicate that SIRT1 overexpression reduces mitochondrial biogenesis (Gurd et al., 2009). This last paper is based on the correlation of SIRT1 levels with mitochondrial biogenesis. In this context, a recent paper indicate that there are a direct relationship between mitochondrial biogenesis and activity of PGC1α with nuclear activity of SIRT1 although not with its protein content in skeletal muscle cells (Gurd et al., 2011) indicating that sirtuins levels are not necessarily related to the activity of these enzymes.

Another of the most studied targets of SIRT1 is the tumor suppressor p53. SIRT1 deacetylates K382 of p53/TP53 and inhibits its transcriptional activity impairing then, its ability to induce proapoptotic mechanisms and to modulate cell senescence. Further, it has been also reported that H2O2-induced cell senescence is accompanied by accumulation of acetylated p53 by decrease in the function of SIRT1 (Furukawa et al., 2007). Taken into consideration the role of p53 in nuclear and mitochondrial apoptosis (Moll & Zaika, 2001), SIRT1 seems to be a p53-dependent antiapoptotic factor.

On the other hand, modulation of p53 by SIRT1 also produces effects on cell metabolism since p53 seems to regulate mitochondrial respiration and glycolysis (Ma et al., 2007). In fact, p53 regulates the transcription of cytochrome c oxidase 2, an important factor in assembly of the cytochrome c oxidase complex (Fields et al., 2007), and then, an important factor in mitochondrial respiration. Then, high levels of SIRT1 in tumor cells will block p53-

**3.3 SIRT3** 

deacetylation.

et al., 2010).

antioxidant systems.

been associated with higher longevity (Falcon et al.).

lysine residues on SOD2 promoting its antioxidant activity.

Sirtuin-Dependent Metabolic Control and Its Role in the Aging Process 105

One of the most important sirtuin in metabolic regulation is SIRT3. Its localization into mitochondria and the diversity of targets including both metabolic and antioxidant components makes it in one of the immediate regulators of mitochondrial activity. In a recent paper, Kendrick and co-authors found that mice fed under high-fat diet develop fatty liver and show high levels of acetylated proteins in parallel with a decrease in SIRT3 activity (Kendrick et al., 2011). Moreover, deletion of SIRT3 further increases acetylation in high-fat fed animals and reduces the activity of respiratory complexes III and IV indicating a key role of this sirtuin in mitochondrial activity control. One of the direct targets of SIRT3 is succinate dehydrogenase (SDH, Complex II). Acetylated SDH show low activity and deacetylation by SIRT3 activates it (Finley et al., 2011). These papers indicate the important role of SIRT3 in the regulation of mitochondrial activities by

The role of SIRT3 in acetate metabolism has been also related to aging (Shimazu et al., 2010). Acetate plays an important role in cell metabolism being an important product of ethanol and fatty acid metabolism especially during fasting or starvation (Seufert et al., 1974). Acetate can be converted into acetyl-CoA by the activity of acetyl-CoA synthase enzymes in cytosol (AceCS1) or mitochondria (AceCS2). These enzymes are activated by deacetylation by both SIRT1 in cytosol and SIRT3 in mitochondria (Shimazu et al., 2010). An important role for SIRT3 in acetate metabolism has been suggested since both, SIRT3 KO and AceCS2 KO mice show overlapping phenotypes. However, to date no clear data about the role of acetate in aging process have been shown although AceCS-mediated synthesis in yeast has

The importance of SIRT3 in the protection against oxidative stress is also important since the protective effect of CR on oxidative stress is diminished in mice lacking SIRT3. This sirtuin is involved in the reduction of cellular ROS levels depending on the manganese-dependent mitochondrial superoxide dismutase 2 (MnSOD or SOD2) (Qiu et al., 2010). SIRT3 adjusts MnSOD activity to the mitochondrial nutrients availability and then, the production of mitochondrial ROS (Ozden et al., 2011). In this regulation, SIRT3 deacetylates two important

Levels of SIRT3 are also regulated by the energetic status of the organism. SIRT3 levels increase by caloric restriction of exposure to low temperatures in brown adipocytes. Forced expression of SIRT3 and activity in a cell line for brown adipocytes enhances the expression of PGC1α, UCP1 and another mitochondria-related genes whereas mutation of SIRT3 inhibits PGC1α-dependent UCP1 expression (Shi et al., 2005). Diet and exercise signals also regulate SIRT3 and activate the AMPK and PGC1α in skeletal muscle cells (Palacios et al., 2009) whereas this activation is much lower in SIRT3 KO animals. On the other hand, PGC1α strongly stimulates mouse SIRT3 gene expression in muscle cells and hepatocytes (Kong et al., 2010) through binding to an oestrogen-related receptor binding element (ERRE) in its promoter region. Induction of SIRT3 is also essential for mitochondrial biogenesis and the expression of several of mitochondrial components including antioxidant systems (Kong

Taken together, SIRT3 seems to be a key sirtuin that senses metabolic status through NAD+/NADH levels at the mitochondria and then, integrates respiratory metabolism and

dependent SCO2 transcription and contribute to the Warburg effect found in these cells. However, the relationship between SIRT1 and p53 is more complex at the transcriptional level. Transcription of SIRT1 is repressed by p53 via p53 response elements in its proximal promoter (Naqvi et al., 2010). However, another p53 binding site has been reported in the distal promoter of SIRT1. This binding site is necessary for SIRT1 induction under caloric restriction (Naqvi et al., 2010). In this site, p53 competes with the Hypermethylated-In-Cancer-1 (HIC1) transcriptional repressor and, then, activation of p53 derepresses SIRT1 transcription. Taken together all the information available about p53 and SIRT1 interaction, more research is be necessary to clarify the complex system p53-SIRT1 and regulation of mitochondrial activity.

All these regulations implicate a contradictory role of SIRT1 in modulation of mitochondrial biogenesis and respiratory metabolism. If SIRT1 is activating PGC1α by deacetylation and, then, inducing mitochondrial biogenesis, downregulation of the activity of p53 by the same SIRT1 will reduce the respiratory capacity by affecting SCO2 levels and Complex IV assembly. This contradictory effect would be explained by the different location of SIRT1 and then, modulation of different regulatory processes. Further, some other studies have indicate that despite the role of SIRT1 as deacetylase of p53, SIRT1 has little effect on p53 dependent transcription and does not affect many of the p53-mediated biological activities (Kamel et al., 2006). If these results are confirmed, they would explain how mitochondrial biogenesis and p53-repression can occur at the same time. Future research will clarify this complex regulation of mitochondrial respiratory metabolism.

#### **3.2 SIRT2**

The human SIRT2 is predominantly a cytosolic protein known to be a tubulin deacetylase (North et al., 2003). SIRT2 deacetylates lysine-40 of α-tubulin and then, its knockdown results in tubulin hyperacetylation. Levels of SIRT2 increases dramatically during mitosis and is also phosphorylated during the G2/M transition. Then, SIRT2 is an important factor in the control of mitotic exit in the cell cycle (Dryden et al., 2003). Further, its interaction with the homeobox transcription factor, HOXA10, raises the possibility that SIRT2 also plays a role in mammalian development (Bae et al., 2004). The importance of tubulin activity in neuronal activity probably explains the important role ascribed to SIRT2 in neurodegenerative diseases (Harting & Knoll, 2010).

Regarding metabolism, information about SIRT2 is very limited and seems to indicate a lipid inhibitory role of this sirtuin in contrast with the role found with SIRT1 and 3. Its presence has been inversely correlated with the differentiation of preadipocytes to adipocytes by modulating the activity of FOXO1 (Jing et al., 2007). SIRT2 deacetylates FOXO1 and enhances its repressive interaction with PPAR-γ, an essential factor in adipocyte differentiation (Wang & Tong, 2009). In contrast with the neuroprotection reported in some neurodegenerative processes, a neuroprotective effect of the decrease of sterol biosynthesis through SIRT2 inhibition has been also shown in the case of Huntington's disease (Luthi-Carter et al., 2010). On the other hand, silencing of SIRT2 induces intracellular ATP drop and cell death in neuronal PC12 cells (Nie et al., 2011) indicating metabolic regulatory mechanisms of this sirtuin. Further, the role of SIRT2 in expression of antioxidant systems has been also reported. Induction of MnSOD seems to depend on deacetylation of FOXOa by SIRT2 (F. Wang et al., 2007).

#### **3.3 SIRT3**

104 Bioenergetics

dependent SCO2 transcription and contribute to the Warburg effect found in these cells. However, the relationship between SIRT1 and p53 is more complex at the transcriptional level. Transcription of SIRT1 is repressed by p53 via p53 response elements in its proximal promoter (Naqvi et al., 2010). However, another p53 binding site has been reported in the distal promoter of SIRT1. This binding site is necessary for SIRT1 induction under caloric restriction (Naqvi et al., 2010). In this site, p53 competes with the Hypermethylated-In-Cancer-1 (HIC1) transcriptional repressor and, then, activation of p53 derepresses SIRT1 transcription. Taken together all the information available about p53 and SIRT1 interaction, more research is be necessary to clarify the complex system p53-SIRT1 and regulation of

All these regulations implicate a contradictory role of SIRT1 in modulation of mitochondrial biogenesis and respiratory metabolism. If SIRT1 is activating PGC1α by deacetylation and, then, inducing mitochondrial biogenesis, downregulation of the activity of p53 by the same SIRT1 will reduce the respiratory capacity by affecting SCO2 levels and Complex IV assembly. This contradictory effect would be explained by the different location of SIRT1 and then, modulation of different regulatory processes. Further, some other studies have indicate that despite the role of SIRT1 as deacetylase of p53, SIRT1 has little effect on p53 dependent transcription and does not affect many of the p53-mediated biological activities (Kamel et al., 2006). If these results are confirmed, they would explain how mitochondrial biogenesis and p53-repression can occur at the same time. Future research will clarify this

The human SIRT2 is predominantly a cytosolic protein known to be a tubulin deacetylase (North et al., 2003). SIRT2 deacetylates lysine-40 of α-tubulin and then, its knockdown results in tubulin hyperacetylation. Levels of SIRT2 increases dramatically during mitosis and is also phosphorylated during the G2/M transition. Then, SIRT2 is an important factor in the control of mitotic exit in the cell cycle (Dryden et al., 2003). Further, its interaction with the homeobox transcription factor, HOXA10, raises the possibility that SIRT2 also plays a role in mammalian development (Bae et al., 2004). The importance of tubulin activity in neuronal activity probably explains the important role ascribed to SIRT2 in

Regarding metabolism, information about SIRT2 is very limited and seems to indicate a lipid inhibitory role of this sirtuin in contrast with the role found with SIRT1 and 3. Its presence has been inversely correlated with the differentiation of preadipocytes to adipocytes by modulating the activity of FOXO1 (Jing et al., 2007). SIRT2 deacetylates FOXO1 and enhances its repressive interaction with PPAR-γ, an essential factor in adipocyte differentiation (Wang & Tong, 2009). In contrast with the neuroprotection reported in some neurodegenerative processes, a neuroprotective effect of the decrease of sterol biosynthesis through SIRT2 inhibition has been also shown in the case of Huntington's disease (Luthi-Carter et al., 2010). On the other hand, silencing of SIRT2 induces intracellular ATP drop and cell death in neuronal PC12 cells (Nie et al., 2011) indicating metabolic regulatory mechanisms of this sirtuin. Further, the role of SIRT2 in expression of antioxidant systems has been also reported. Induction of MnSOD seems to depend on deacetylation of FOXOa

complex regulation of mitochondrial respiratory metabolism.

neurodegenerative diseases (Harting & Knoll, 2010).

by SIRT2 (F. Wang et al., 2007).

mitochondrial activity.

**3.2 SIRT2** 

One of the most important sirtuin in metabolic regulation is SIRT3. Its localization into mitochondria and the diversity of targets including both metabolic and antioxidant components makes it in one of the immediate regulators of mitochondrial activity. In a recent paper, Kendrick and co-authors found that mice fed under high-fat diet develop fatty liver and show high levels of acetylated proteins in parallel with a decrease in SIRT3 activity (Kendrick et al., 2011). Moreover, deletion of SIRT3 further increases acetylation in high-fat fed animals and reduces the activity of respiratory complexes III and IV indicating a key role of this sirtuin in mitochondrial activity control. One of the direct targets of SIRT3 is succinate dehydrogenase (SDH, Complex II). Acetylated SDH show low activity and deacetylation by SIRT3 activates it (Finley et al., 2011). These papers indicate the important role of SIRT3 in the regulation of mitochondrial activities by deacetylation.

The role of SIRT3 in acetate metabolism has been also related to aging (Shimazu et al., 2010). Acetate plays an important role in cell metabolism being an important product of ethanol and fatty acid metabolism especially during fasting or starvation (Seufert et al., 1974). Acetate can be converted into acetyl-CoA by the activity of acetyl-CoA synthase enzymes in cytosol (AceCS1) or mitochondria (AceCS2). These enzymes are activated by deacetylation by both SIRT1 in cytosol and SIRT3 in mitochondria (Shimazu et al., 2010). An important role for SIRT3 in acetate metabolism has been suggested since both, SIRT3 KO and AceCS2 KO mice show overlapping phenotypes. However, to date no clear data about the role of acetate in aging process have been shown although AceCS-mediated synthesis in yeast has been associated with higher longevity (Falcon et al.).

The importance of SIRT3 in the protection against oxidative stress is also important since the protective effect of CR on oxidative stress is diminished in mice lacking SIRT3. This sirtuin is involved in the reduction of cellular ROS levels depending on the manganese-dependent mitochondrial superoxide dismutase 2 (MnSOD or SOD2) (Qiu et al., 2010). SIRT3 adjusts MnSOD activity to the mitochondrial nutrients availability and then, the production of mitochondrial ROS (Ozden et al., 2011). In this regulation, SIRT3 deacetylates two important lysine residues on SOD2 promoting its antioxidant activity.

Levels of SIRT3 are also regulated by the energetic status of the organism. SIRT3 levels increase by caloric restriction of exposure to low temperatures in brown adipocytes. Forced expression of SIRT3 and activity in a cell line for brown adipocytes enhances the expression of PGC1α, UCP1 and another mitochondria-related genes whereas mutation of SIRT3 inhibits PGC1α-dependent UCP1 expression (Shi et al., 2005). Diet and exercise signals also regulate SIRT3 and activate the AMPK and PGC1α in skeletal muscle cells (Palacios et al., 2009) whereas this activation is much lower in SIRT3 KO animals. On the other hand, PGC1α strongly stimulates mouse SIRT3 gene expression in muscle cells and hepatocytes (Kong et al., 2010) through binding to an oestrogen-related receptor binding element (ERRE) in its promoter region. Induction of SIRT3 is also essential for mitochondrial biogenesis and the expression of several of mitochondrial components including antioxidant systems (Kong et al., 2010).

Taken together, SIRT3 seems to be a key sirtuin that senses metabolic status through NAD+/NADH levels at the mitochondria and then, integrates respiratory metabolism and antioxidant systems.

Sirtuin-Dependent Metabolic Control and Its Role in the Aging Process 107

proinflammatory processes, apoptosis and cellular senescence (Kawahara et al., 2009). Then, the control of gene expression by histone modulation seems to be a key factor in SIRT6-

Furthermore, the activity of SIRT6 in liver has been also reported. Rosiglitazone (RGZ) is used to protect liver against steatosis. This compound increases the levels of SIRT6 in liver at the same time that ameliorates hepatic liver accumulation affecting PGC1α and FOXO1 (Yang et al., 2011). However, in SIRT6-deficient mice, RGZ was unable to decrease fat accumulation in hepatocytes and to affect PGC1α and FOXO1 activity indicating an important role of this sirtuin in fat storage in liver. In this mechanism, SIRT1 could be also involved since it forms a complex with FOXO3 and NRF1 and activates the expression of SIRT6 (Kim et al., 2010). In this case SIRT6 would be the sirtuin that negatively regulates glycolysis, triglyceride synthesis and fat metabolism by deacetylating H3K9 and then, modifying the activity of the promoters of many genes involved in metabolic processes. In fact, the specific deletion of SIRT6 in liver causes profound alterations in gene expression that produce the contrary effects in glycolysis, triglyceride synthesis and fat metabolism. The last member of sirtuins, SIRT7, is widely expressed in nucleolus and has been associated with active rRNA genes interacting with RNA polymerase I and with histones (Ford et al., 2006). This sirtuin is controlled by CDK1-cyclin B-dependent phosphorylation and dephosphorylation indicating that its activity is required to resume rDNA transcription in late telophase (Grob et al., 2009). In the case of SIRT7, studies performed by using murine cells lacking or overexpressing this sirtuin demonstrate that it is related with the tumorigenic potential and may enable cells to sustain critical metabolic functions because it inhibits cell growth under severe stress conditions (Vakhrusheva, Braeuer et al., 2008). These studies have also demonstrated the important role of this sirtuin in lifespan. In fact, mice lacking SIRT7 undergo reduction in mean and maximum lifespans and develop heart hypertrophy and inflammatory cardiopathy (Vakhrusheva, Smolka et al., 2008) probably by

the impossibility to deacetylate p53 and regulate p53-dependent apoptosis.

antioxidant systems such as SOD2 in mitochondria by SIRT3 (Qiu et al., 2010).

to the decrease in the expression of SIRT1 (Ferrara et al., 2008).

Other of the important roles of sirtuins related to metabolism and aging is based on their activity to maintain cellular antioxidant mechanisms and autophagy systems. A great body of evidence has accumulated indicating that at the same time that sirtuins are modulating metabolism, they also regulate, in a coordinated mechanism, antioxidant systems and

Altered ROS levels are observed in several age-related illnesses including carcinogenesis, neurodegenerative, fatty liver, insulin resistance, cardiac resistance, etc. In mitochondria MnSOD is the primary ROS scavenging enzyme to converts superoxide to hydrogen peroxide that is further converted to water by catalase and other peroxidases. In this mechanism SIRT3 exert a key role since changes in lysine acetylation modifies MnSOD activity in mitochondria (Ozden et al., 2011). Further, CR effect depends, at least in part, on sirtuin regulation but at the same time oxidative stress is reduced in CR by activation of

In heart, the aging-related decrease in MnSOD and catalase expression accompanied by increase in the levels of oxidative damage indicated by TBARS and 4-HNE has been related

**4. Sirtuins: Antioxidant mechanisms and autophagy** 

dependent prolongevity effect.

recycling systems in cells.

#### **3.4 SIRT4 and SIRT5**

SIRT4 and SIRT5 are also mitochondrial sirtuins involved in the regulation of other metabolic processes essentially related with the urea cycle (Li & Kazgan, 2011). One of the main activities of SIRT4 in mitochondria is the downregulation of insulin secretion by beta cells by repressing the activity of glutamate dehydrogenase in response to aminoacids (Argmann & Auwerx, 2006; Haigis et al., 2006). Depletion of SIRT4 have also shown that this sirtuin seems to exert an opposite role than SIRT1 and SIRT3 since its depletion increases gene expression of fatty acid metabolism enzymes in hepatocytes (Nasrin et al., 2010). However, this effect is indirect due to compensatory mechanisms involving higher expression of SIRT1. However, putative contrary effects of this sirtuin in relationship with other members of the family cannot be discarded and further research involving specific inhibitors instead of gene depletion is needed.

In the case of SIRT5, this sirtuin, mainly located at the matrix of the mitochondria, is also involved in the regulation of the urea cycle (Nakagawa & Guarente, 2009). SIRT5 deacetylates the mitochondrial carbamoyl phosphate synthetase 1. This enzyme is the first and rate-limiting step of the urea cycle (Nakagawa & Guarente, 2009). Furthermore, deacetylation of cytochrome c has been also reported although the effect of this deacetylation in both, respiration or apoptosis, is not clear (Schlicker et al., 2008). To date, no other substrates of SIRT5 have been reported.

#### **3.5 SIRT6 and SIRT7**

SIRT6 is predominantly a nuclear protein broadly expressed in tissues showing the highest levels in muscle, brain and heart (Liszt et al., 2005). In any case, SIRT6 is mainly involved in DNA damage repair and is located in the nucleus. It is recruited to the sites of DNA doublestrand breaks (DSBs) and stimulates DSB repair through both, nonhomologous end joining and homologous recombination by stimulating PARP-1 poly-ADP-rybosilase activity (Mao et al., 2011).

It seems that this sirtuins is closely involved in neural degeneration related to aging. In fact, a mice model lacking SIRT6 develops a degenerative disorder that mimics models of accelerated aging (Lombard et al., 2008, Mostoslavsky et al., 2006). This effect depends on a higher instability through the DNA base excision repair pathway, then, the accumulation of mutations in the genome leads to aging-associated degenerative phenotypes (Mostoslavsky et al., 2006). Furthermore, SIRT6-deficient mice show deficiency in growth and show severe metabolic defects indicating that the higher DNA-damage found in these animals is linked to a systemic metabolic deregulation that leads to age-related processes and death.

Neural SIRT6 has been also recently related to metabolic homeostasis in mammals. Neuralspecific deletion of SIRT6 in mice produces postnatal growth retardation since these animals show low growth hormone (GH) and also insulin-like growth factor 1 (IGF1) levels (Schwer et al., 2010). However, unlike SIRT6-KO animals that die by hypoglycaemia and other severe metabolic defects (Mostoslavsky et al., 2006), neural-SIRT6 KO animals, reach normal size and even become obese. It seems that at the central nervous system, SIRT6 acts as a central regulator of somatic growth and metabolism by modulating neuroendocrine system. It seems that the main mechanism of action of SIRT6 in the regulation of gene expression and the control or systemic metabolism and aging is through the deacetylation of lysine 9 in histone H3. Recently it has been shown that hyperacetylation of H3K9 found in SIRT6 deficient cells leads to a higher NF-κB-dependent modulation of gene expression,

SIRT4 and SIRT5 are also mitochondrial sirtuins involved in the regulation of other metabolic processes essentially related with the urea cycle (Li & Kazgan, 2011). One of the main activities of SIRT4 in mitochondria is the downregulation of insulin secretion by beta cells by repressing the activity of glutamate dehydrogenase in response to aminoacids (Argmann & Auwerx, 2006; Haigis et al., 2006). Depletion of SIRT4 have also shown that this sirtuin seems to exert an opposite role than SIRT1 and SIRT3 since its depletion increases gene expression of fatty acid metabolism enzymes in hepatocytes (Nasrin et al., 2010). However, this effect is indirect due to compensatory mechanisms involving higher expression of SIRT1. However, putative contrary effects of this sirtuin in relationship with other members of the family cannot be discarded and further research involving specific

In the case of SIRT5, this sirtuin, mainly located at the matrix of the mitochondria, is also involved in the regulation of the urea cycle (Nakagawa & Guarente, 2009). SIRT5 deacetylates the mitochondrial carbamoyl phosphate synthetase 1. This enzyme is the first and rate-limiting step of the urea cycle (Nakagawa & Guarente, 2009). Furthermore, deacetylation of cytochrome c has been also reported although the effect of this deacetylation in both, respiration or apoptosis, is not clear (Schlicker et al., 2008). To date, no

SIRT6 is predominantly a nuclear protein broadly expressed in tissues showing the highest levels in muscle, brain and heart (Liszt et al., 2005). In any case, SIRT6 is mainly involved in DNA damage repair and is located in the nucleus. It is recruited to the sites of DNA doublestrand breaks (DSBs) and stimulates DSB repair through both, nonhomologous end joining and homologous recombination by stimulating PARP-1 poly-ADP-rybosilase activity (Mao

It seems that this sirtuins is closely involved in neural degeneration related to aging. In fact, a mice model lacking SIRT6 develops a degenerative disorder that mimics models of accelerated aging (Lombard et al., 2008, Mostoslavsky et al., 2006). This effect depends on a higher instability through the DNA base excision repair pathway, then, the accumulation of mutations in the genome leads to aging-associated degenerative phenotypes (Mostoslavsky et al., 2006). Furthermore, SIRT6-deficient mice show deficiency in growth and show severe metabolic defects indicating that the higher DNA-damage found in these animals is linked

Neural SIRT6 has been also recently related to metabolic homeostasis in mammals. Neuralspecific deletion of SIRT6 in mice produces postnatal growth retardation since these animals show low growth hormone (GH) and also insulin-like growth factor 1 (IGF1) levels (Schwer et al., 2010). However, unlike SIRT6-KO animals that die by hypoglycaemia and other severe metabolic defects (Mostoslavsky et al., 2006), neural-SIRT6 KO animals, reach normal size and even become obese. It seems that at the central nervous system, SIRT6 acts as a central regulator of somatic growth and metabolism by modulating neuroendocrine system. It seems that the main mechanism of action of SIRT6 in the regulation of gene expression and the control or systemic metabolism and aging is through the deacetylation of lysine 9 in histone H3. Recently it has been shown that hyperacetylation of H3K9 found in SIRT6 deficient cells leads to a higher NF-κB-dependent modulation of gene expression,

to a systemic metabolic deregulation that leads to age-related processes and death.

**3.4 SIRT4 and SIRT5** 

**3.5 SIRT6 and SIRT7** 

et al., 2011).

inhibitors instead of gene depletion is needed.

other substrates of SIRT5 have been reported.

proinflammatory processes, apoptosis and cellular senescence (Kawahara et al., 2009). Then, the control of gene expression by histone modulation seems to be a key factor in SIRT6 dependent prolongevity effect.

Furthermore, the activity of SIRT6 in liver has been also reported. Rosiglitazone (RGZ) is used to protect liver against steatosis. This compound increases the levels of SIRT6 in liver at the same time that ameliorates hepatic liver accumulation affecting PGC1α and FOXO1 (Yang et al., 2011). However, in SIRT6-deficient mice, RGZ was unable to decrease fat accumulation in hepatocytes and to affect PGC1α and FOXO1 activity indicating an important role of this sirtuin in fat storage in liver. In this mechanism, SIRT1 could be also involved since it forms a complex with FOXO3 and NRF1 and activates the expression of SIRT6 (Kim et al., 2010). In this case SIRT6 would be the sirtuin that negatively regulates glycolysis, triglyceride synthesis and fat metabolism by deacetylating H3K9 and then, modifying the activity of the promoters of many genes involved in metabolic processes. In fact, the specific deletion of SIRT6 in liver causes profound alterations in gene expression that produce the contrary effects in glycolysis, triglyceride synthesis and fat metabolism.

The last member of sirtuins, SIRT7, is widely expressed in nucleolus and has been associated with active rRNA genes interacting with RNA polymerase I and with histones (Ford et al., 2006). This sirtuin is controlled by CDK1-cyclin B-dependent phosphorylation and dephosphorylation indicating that its activity is required to resume rDNA transcription in late telophase (Grob et al., 2009). In the case of SIRT7, studies performed by using murine cells lacking or overexpressing this sirtuin demonstrate that it is related with the tumorigenic potential and may enable cells to sustain critical metabolic functions because it inhibits cell growth under severe stress conditions (Vakhrusheva, Braeuer et al., 2008). These studies have also demonstrated the important role of this sirtuin in lifespan. In fact, mice lacking SIRT7 undergo reduction in mean and maximum lifespans and develop heart hypertrophy and inflammatory cardiopathy (Vakhrusheva, Smolka et al., 2008) probably by the impossibility to deacetylate p53 and regulate p53-dependent apoptosis.
