**1. Introduction**

94 Bioenergetics

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and PKR in UVA-induced signal transduction toward phosphorylation of eIF2

During last years, the protein family of sirtuins, composed by NAD+-dependent deacetylases, has emerged as a key factor in aging. From yeast to humans, sirtuins are involved in metabolic changes that induce a higher respiratory capacity accompanied by lower oxidative damage. They are involved in the control of glucose catabolism, fatty acid metabolism, respiratory chain activity in mitochondria and several other metabolic processes including control of antioxidant capacity in cells and tissues (Dali-Youcef et al., 2007; Elliott & Jirousek, 2008; Lomb et al., 2010; Pallas et al., 2008).

As these deacetylases are dependent on the NAD+/NADH ratio, they can be considered as important sensors of the metabolic status of the cells and probably because this they are one of the main family of proteins involved in the regulation of metabolism in the cell (Li & Kazgan, 2011). Further, their relationship with the AMPK-dependent pathway, that controls respiratory metabolism by inhibiting insulin-dependent signaling, highlights the importance of these proteins in metabolic regulation and especially in insulin-resistance, diabetes and obesity (Canto et al., 2009; Ruderman et al., 2010).

Sirtuins have been involved in aging process and considered important factors in delaying aging process and increase longevity (Guarente, 2000; Tissenbaum & Guarente, 2001). However, very recent studies have questioned the role of these deacetylases in longevity (Burnett et al., 2011; Viswanathan & Guarente, 2011). But their activity in yeast, worms and flies still permits to correlate its function in metabolism and dietarydependent modulations with aging process (Guarente, 2008). However, to date, in mammals and, especially in humans, their role in longevity is not clear. Whereas in lower organisms only one member has been found, SIR2, in mammals, seven members have been described to date. This fact indicates a higher complexity in interactions, targets and functions in higher animals than in lowers. Further, in mammals, the specific distribution of these deacetylases among the different cell compartments also indicates several localdependent influences of sirtuins.

<sup>\*</sup> Corresponding Author

Sirtuin-Dependent Metabolic Control and Its Role in the Aging Process 97

The first member of this family studied in deep was the yeast Sir2. This deacetylase is responsible of silencing chromatin by deacetylation of histones (Blander & Guarente, 2004) and has been related to the increase in longevity in yeast, worms and flies. Apart of its activity reducing the accumulation of chromatin of ribosomal RNA (rRNA) genes in yeast, the prolongevity of Sir2 has been also related to the modulation of mitochondrial function

To date, seven sirtuins have been described in mammals. They are designed as SIRT1 through SIRT7. Based on the homology of the 250 aminoacids core domain, the mitochondrial SIRT3, the nuclear-cytosolic SIRT1 and the mainly nuclear SIRT2 show the closest homology to yeast SIR2 (Frye, 2000). However, if we attend to the alignment of the aminoacid sequence of the human members we can see that the identity at the aminoacid sequence is very low among the members of sirtuins family (Table 1) and only deacetylase sirtuin-type domain shows some homology being highly in the NAD+-binding and in the catalytic domains (Figure 2). A possible explanation for these high differences in sequence between the members of sirtuin family in mammals can be found in the plethora of targets that can be recognized by the different members of the family and in their different and

hSIRT1 hSIRT2 hSIRT3 hSIRT4 hSIRT5 hSIRT6 hSIRT7

Fig. 1. Deacetylation of K-acetyl residues in targets of sirtuins.

selective locations into the cell.

hSIRT1 100

hSIRT2 34.34 100

hSIRT3 30.94 44.85 100

hSIRT4 24.55 21.88 26.02 100

hSIRT5 25.63 22.71 27.66 27.13 100

hSIRT6 21.56 24.21 28.84 28.43 20.59 100

hSIRT7 20.97 22.28 23.70 28.14 22.01 36.77 100 Table 1. Pairwise comparison of aminoacid sequences from the human sirtuin members. From BLAST (basic local alignment search tool) analysis of the indicated proteins in figure 2.

providing benefit to slow aging and associated diseases (Guarente, 2008).

Aging can be considered a severe deleterious process that affects all the compartments in cells and also all the tissues and organs in the organism. Apart of the different theories of aging (Jin, 2010), the main common factor is the accumulation of non-metabolizable or degradable molecules into cells and tissues that impair their correct function. In few words, we age because our organism accumulates rubbish and we are unable to eliminate or recycle it. Most of the damaged molecules are the result of a unbalanced metabolism that produces high levels of reactive molecules accompanied by a low capacity of the endogenous antioxidant mechanisms of cells and the recycling mechanisms such as proteasome and autophagy or DNA-damage reparation (Asha Devi, 2009; Fleming & Bensch, 1991; Maynard et al., 2009; Perez et al., 2009; Sohal et al., 1994). As results, oxidized molecules accumulate into cells impairing their physiology at all levels. Then, a balanced and controlled metabolism will improve oxidant/antioxidant relationship and delay the accumulation of oxidized molecules in aged cells and tissues.

The present chapter is focused on the role of the metabolism in aging process and the importance of sirtuins in its control. We will describe the different pathways regulated by sirtuins and how modifications in NAD+/NADH ratio can affect the activity of these deacetylases. Moreover, we will discuss the possible role of NADH-dependent oxidoreductases in the control of metabolism through these proteins. Furthermore, the role of a known polyphenol, resveratrol, as agonist of sirtuins and caloric restriction in aging and metabolic control will be also revised.

#### **2. Sirtuins, a heterogeneous family of protein deacetylases**

Sirtuins are a family of proteins that share a conserved NAD+-dependent acetyl-lysine deacetylase and ADP-ribosyltransferase activity. They have been related to the regulation of the metabolism and also lifespan being involved in cell survival and apoptosis, cell proliferation and senescence. They are widely located in all the organs and near all the subcellular locations. The seven isoforms found to date in humans localize either in the nucleus, cytoplasm or mitochondria. The use of modified organisms showing increasing gene dosage of sirtuin orthologs in eukaryotes such as yeast, worms or flies have demonstrated that these enzymes are directly involved in lengthening of longevity (Guarente, 2007). Further, the relationship between calorie restriction and longevity indicate that metabolism is directly involved in aging, and then, as sirtuins are involved in the control of metabolism, a direct link between the activity and modulation of these proteins and a longer lifespan seems to be convincing (Balcerczyk & Pirola, 2010).

In contrast with class I, II and IV deacetylases, mainly involved in the control of epigenetic processes (Kuzmichev & Reinberg, 2001), sirtuins are members of the class III characterized to be dependent on NAD+. These enzymes catalyze the reaction shown in figure 1. They bind to a Nε-acetyl-lysines of the target protein and deacetylate them by using NAD+ as substrate and producing nicotinamide (NAM) and 2'-O-acetyl-ribose (2'-O-AADPR) as products (Hirsch & Zheng, 2011). In this process, increasing levels of NAD+ increase the activity of sirtuins whereas higher NAM or NADH levels exert an inhibitory effect (Wolberger, 2007). Further, the expression of sirtuins are also regulated by the ratio NAD+/NADH since higher mRNA levels have been found when NADH/NAD+ levels rise (Gambini et al., 2011). Then, they can be considered as metabolic sensors since they can modulate their activity and levels depending on the ratio NAD+/NADH.

Aging can be considered a severe deleterious process that affects all the compartments in cells and also all the tissues and organs in the organism. Apart of the different theories of aging (Jin, 2010), the main common factor is the accumulation of non-metabolizable or degradable molecules into cells and tissues that impair their correct function. In few words, we age because our organism accumulates rubbish and we are unable to eliminate or recycle it. Most of the damaged molecules are the result of a unbalanced metabolism that produces high levels of reactive molecules accompanied by a low capacity of the endogenous antioxidant mechanisms of cells and the recycling mechanisms such as proteasome and autophagy or DNA-damage reparation (Asha Devi, 2009; Fleming & Bensch, 1991; Maynard et al., 2009; Perez et al., 2009; Sohal et al., 1994). As results, oxidized molecules accumulate into cells impairing their physiology at all levels. Then, a balanced and controlled metabolism will improve oxidant/antioxidant relationship and delay the accumulation of

The present chapter is focused on the role of the metabolism in aging process and the importance of sirtuins in its control. We will describe the different pathways regulated by sirtuins and how modifications in NAD+/NADH ratio can affect the activity of these deacetylases. Moreover, we will discuss the possible role of NADH-dependent oxidoreductases in the control of metabolism through these proteins. Furthermore, the role of a known polyphenol, resveratrol, as agonist of sirtuins and caloric restriction in aging and

Sirtuins are a family of proteins that share a conserved NAD+-dependent acetyl-lysine deacetylase and ADP-ribosyltransferase activity. They have been related to the regulation of the metabolism and also lifespan being involved in cell survival and apoptosis, cell proliferation and senescence. They are widely located in all the organs and near all the subcellular locations. The seven isoforms found to date in humans localize either in the nucleus, cytoplasm or mitochondria. The use of modified organisms showing increasing gene dosage of sirtuin orthologs in eukaryotes such as yeast, worms or flies have demonstrated that these enzymes are directly involved in lengthening of longevity (Guarente, 2007). Further, the relationship between calorie restriction and longevity indicate that metabolism is directly involved in aging, and then, as sirtuins are involved in the control of metabolism, a direct link between the activity and modulation of these proteins

In contrast with class I, II and IV deacetylases, mainly involved in the control of epigenetic processes (Kuzmichev & Reinberg, 2001), sirtuins are members of the class III characterized to be dependent on NAD+. These enzymes catalyze the reaction shown in figure 1. They bind to a Nε-acetyl-lysines of the target protein and deacetylate them by using NAD+ as substrate and producing nicotinamide (NAM) and 2'-O-acetyl-ribose (2'-O-AADPR) as products (Hirsch & Zheng, 2011). In this process, increasing levels of NAD+ increase the activity of sirtuins whereas higher NAM or NADH levels exert an inhibitory effect (Wolberger, 2007). Further, the expression of sirtuins are also regulated by the ratio NAD+/NADH since higher mRNA levels have been found when NADH/NAD+ levels rise (Gambini et al., 2011). Then, they can be considered as metabolic sensors since they can

**2. Sirtuins, a heterogeneous family of protein deacetylases** 

and a longer lifespan seems to be convincing (Balcerczyk & Pirola, 2010).

modulate their activity and levels depending on the ratio NAD+/NADH.

oxidized molecules in aged cells and tissues.

metabolic control will be also revised.

Fig. 1. Deacetylation of K-acetyl residues in targets of sirtuins.

The first member of this family studied in deep was the yeast Sir2. This deacetylase is responsible of silencing chromatin by deacetylation of histones (Blander & Guarente, 2004) and has been related to the increase in longevity in yeast, worms and flies. Apart of its activity reducing the accumulation of chromatin of ribosomal RNA (rRNA) genes in yeast, the prolongevity of Sir2 has been also related to the modulation of mitochondrial function providing benefit to slow aging and associated diseases (Guarente, 2008).

To date, seven sirtuins have been described in mammals. They are designed as SIRT1 through SIRT7. Based on the homology of the 250 aminoacids core domain, the mitochondrial SIRT3, the nuclear-cytosolic SIRT1 and the mainly nuclear SIRT2 show the closest homology to yeast SIR2 (Frye, 2000). However, if we attend to the alignment of the aminoacid sequence of the human members we can see that the identity at the aminoacid sequence is very low among the members of sirtuins family (Table 1) and only deacetylase sirtuin-type domain shows some homology being highly in the NAD+-binding and in the catalytic domains (Figure 2). A possible explanation for these high differences in sequence between the members of sirtuin family in mammals can be found in the plethora of targets that can be recognized by the different members of the family and in their different and selective locations into the cell.


Table 1. Pairwise comparison of aminoacid sequences from the human sirtuin members. From BLAST (basic local alignment search tool) analysis of the indicated proteins in figure 2.


Sirtuin-Dependent Metabolic Control and Its Role in the Aging Process 99

+ binding domains are

Fig. 2. Alignment of centre core of human sirtuins family in comparison with yeast sir2 (previous page). The figure represent Clustalw alignment from indicated yeast and human sirtuins indicated by their UniProtKB accession numbers. In yeast sir2, the deacetylase sirtuin-type dominium is from 245 to 529 (red arrow) that correspond with the highest

indicated in green, there are the most conserved domains in the whole family. The active site is determined by a histidine at 364 position of sir2 that acts as a proton acceptor, the key histidines in other members have been determined in silico by homology. Although it has been indicated that these enzymes do not bind zinc, probable cystein residues able to bind

regulation, in sir2 two points of regulation by phosphorylation, phosphorylation at serine 23 and at tyrosine 400 have been determined (violet residues). None of them are conserved residues in human sirt forms. Further, in SIRT1, modifications at cysteines 395 and 398 by s-

SIR2 100 41 43 39 45 40 40

Among the other human sirtuins, SIRT4 and SIRT5 are mitochondrial sirtuins that show predominant ADP-ribosyl-transferase activity and a weak deacetylase activity and are involved in urea cycle regulation (Nakagawa & Guarente, 2009). On the other hand, SIRT6 and SIRT7 are considered as members of another subclass of sirtuins involved in reparation of DNA and the control of ribosomal RNA production through cell cycle (Lombard et al., 2008). Although it has been described that sirtuins does not bind zinc, Sir2, SIRT1, -2 and 3 share four proximal cysteines that can indicate the possibility of binding zinc (figure 2). These four Cys are highly conserved among these sirtuins and just following the catalytic histidine. Then, although this Zinc ion must be not involved in the catalytic activity, their presence can be important for the maintenance of the structure of the sirtuin. In fact, recently Sanders and coworkers (Sanders et al., 2010) have shown that the four-cysteine metal binding site resembles the Zn-ribbon structure of transcription factors such as TF-IIS, TF-IIN and RNA polymerase II subunit RPB9. Further, although the Zinc-binding site is too far from catalytic domain, its presence is important for the activity of the enzyme since the change of any cystein to alanine or addition of zinc chelators inhibits the *in vitro* deacetylase activity of

Regarding post-translational regulatory mechanisms, sirtuins can be regulated by phosphorylation and sumoylation. In fact, in SIRT1 thirteen residues have been found to be phosphorylated *in vivo* (Sasaki et al., 2008) indicating a high ratio of regulation by kinases. Further, dephosphorylation by protein phosphatases *in vitro* results in the decrease of the

+-dependent deacetylase activity in SIRT1. On the other hand, sumoylation of SIRT1 at

Table 2. Pairwise comparison of the aminoacid sequences among yeast (*Saccharomyces cerevisiae*) SIR2 and higher homologues in model animals: fission yeast (Saccharomyces pombe); fly (*Drosophila melanogaster*); worm (*Caenorhabditis elegans*); zebrafish (*Dario rerio*); mice (*Mus musculus*) and human (*Homo sapiens*). The percentage of identity in comparison with *S. cerevisiae* sir2 protein is indicated. From BLAST analysis of the indicated proteins.

C. elegans SIR2

D. rerio SIR2

M. musc. SIRT1

H. sapiens SIRT1

zinc are also conserved in some of the members of the family (in blue). Regarding

nitrosylation impede the binding of NAD+ and then, the activity of the enzyme.

D. melanog. SIR2.1

homology sequence among the members of the family. The NAD

S. cerev. SIR2

S. cerev.

sirtuins (Min et al., 2001).

NAD

S. pombe SIR2

**ySIR2/P06700 NPSNGIFYGPSFTKRESLNARMFLKYYG------AHKFLDTYLPEDLNSLYIYYLIKLLGFEVKDQALIGTINSIVHINSQERVQDLGSAISVTNVEDPLAKKQTVRLIKDLQRAINKVL 232 hSIRT1/Q96EB6 NLYDEDDDDEGEEEEEAAAAAIGYRDNLLFGDEIITNGFHSCESDEEDRASHASSSDWTPRPRIGPYTFVQQHLMIGTDPRTILKDLLPETIPPPELDDMTLWQIVINILSEPPKR---- 234 hSIRT2/Q8IXJ6 NLFSQTLSLGSQKER--------------------------------------------------------------------------------LLDELTLEGVARYMQSE-------- 74 hSIRT3/Q9NTG7 GASSVVGSGGSSDK-----------------------------------------------------------------------------------GKLSLQDVAELIRAR-------- 135 hSIRT4/Q9Y6E7 PASPP-------------------------------------------------------------------------------------------LDPEKVKELQRFITLS-------- 54 hSIRT5/Q9NXA8 ARPSS-----------------------------------------------------------------------------------------------SMADFRKFFAKA-------- 50 hSIRT6/Q8N6T7 DPPEE--------------------------------------------------------------------------------------------LERKVWELARLVWQS-------- 44 hSIRT7/Q9NRC8 DDPEE--------------------------------------------------------------------------------------------LRGKVRELASAVRNA-------- 99 . ySIR2/P06700 CTRLRLSNFFTIDHFIQKLHTARKILVLTGAGVSTSLGIPDFRS-SEGFYS--KIKHLGLDDPQDVFNYNIFMHDPSVFYNIANMVLPPEKIYSPLHSFIKMLQMKGKLLRNYTQNIDNL 349 hSIRT1/Q96EB6 ---KKRKDINTIEDAVKLLQECKKIIVLTGAGVSVSCGIPDFRS-RDGIYARLAVDFPDLPDPQAMFDIEYFRKDPRPFFKFAKEIYPGQFQPSLCHKFIALSDKEGKLLRNYTQNIDTL 350 hSIRT2/Q8IXJ6 --------------------RCRRVICLVGAGISTSAGIPDFRSPSTGLYD--NLEKYHLPYPEAIFEISYFKKHPEPFFALAKELYPGQFKPTICHYFMRLLKDKGLLLRCYTQNIDTL 172 hSIRT3/Q9NTG7 --------------------ACQRVVVMVGAGISTPSGIPDFRSPGSGLYS--NLQQYDLPYPEAIFELPFFFHNPKPFFTLAKELYPGNYKPNVTHYFLRLLHDKGLLLRLYTQNIDGL 233 hSIRT4/Q9Y6E7 ----------------------KRLLVMTGAGISTESGIPDYRSEKVGLYA----RTDRRPIQHGDFVRSAPIRQRYWARNFVGWPQFSSHQPNPAHWALSTWEKLGKLYWLVTQNVDAL 148 hSIRT5/Q9NXA8 ----------------------KHIVIISGAGVSAESGVPTFRG-AGGYWR--KWQAQDLATPLAFAHNPSRVWEFYHYRREVMGSKEPNAGHRAIAECETRLGKQGRRVVVITQNIDEL 145 hSIRT6/Q8N6T7 ----------------------SSVVFHTGAGISTASGIPDFRG------------------PHGVWTMEERGLAPKFDTTFES------ARPTQTHMALVQLERVGLLRFLVSQNVDGL 118 hSIRT7/Q9NRC8 ----------------------KYLVVYTGAGISTAASIPDYRG------------------PNGVWTLLQKGRS-------VSAADLSEAEPTLTHMSITRLHEQKLVQHVVSQNCDGL 721 :: \*\*\*:\*. .:\*::\*. :\*\* \* \* ySIR2/P06700 ESYAGISTDKLVQCHGSFATATCV--TCHWNLPGERIFNKIRNLELPLCPYCYKKRREYFPEGYNNKVGVAASQGSMSERPPYILNSYGVLKPDITFFGEA---LPNKFHKSIREDILEC 464 hSIRT1/Q96EB6 EQVAGIQR--IIQCHGSFATASCL--ICKYKVDCEAVRGDIFNQVVPRCPRCPAD------------------------------EPLAIMKPEIVFFGEN---LPEQFHRAMKYDKDEV 433 hSIRT2/Q8IXJ6 ERIAGLEQEDLVEAHGTFYTSHCVSASCRHEYPLSWMKEKIFSEVTPKCEDCQS-----------------------------------LVKPDIVFFGES---LPARFFSCMQSDFLKV 254 hSIRT3/Q9NTG7 ERVSGIPASKLVEAHGTFASATCT--VCQRPFPGEDIRADVMADRVPRCPVCTG-----------------------------------VVKPDIVFFGEP---LPQRFLLHVV-DFPMA 312 hSIRT4/Q9Y6E7 HTKAGSRR--LTELHGCMDRVLCL--DCGEQTPRGVLQERFQVLNPTWSAEAHGLAPD--------GDVFLSEEQVRSFQVPTCVQCGGHLKPDVVFFGDT---VNPDKVDFVHKRVKEA 253 hSIRT5/Q9NXA8 HRKAGTKN--LLEIHGSLFKTRCTSCGVVAENYKSPICPALSGKGAPEPGTQDASIP------------------VEKLPRCEEAGCGGLLRPHVVWFGEN---LDPAILEEVDRELAHC 242 hSIRT6/Q8N6T7 HVRSGFPRDKLAELHGNMFVEECA--KCKTQYVRDTVVGTMGLKATGRLCTVAKARG--------------------------LRACRGELRDTILDWEDS---LPDRDLALADEASRNA 207 hSIRT7/Q9NRC8 HLRSGLPRTAISELHGNMYIEVCT--SCVPNREYVRVFDVTERTALHRHQTGRTCHK-----------------------------CGTQLRDTIVHFGERGTLGQPLNWEAATEAASRA 261 . :\* : : \*\* : \* : : :: : : : ySIR2/P06700 DLLICIGTSLKVAP-VSEIVNMVPSHVPQVLINRD---------------------------------------------PVKHAEFDLSLLGYCDDI---------------------A 517 hSIRT1/Q96EB6 DLLIVIGSSLKVRP-VALIPSSIPHEVPQILINREPLPHLHFDVELLGDCDVIINELCHRLGGEYAKLCCNPVKLSEITEKPPRTQKELAYLSELPPTPLHVSEDSSSPERTSPPDSSVI 552 hSIRT2/Q8IXJ6 DLLLVMGTSLQVQP-FASLISKAPLSTPRLLINKE------------------------KAGQSDPFLGMIMGLGGGMDFDSKKAYRDVAWLGECDQG---------------------C 328 hSIRT3/Q9NTG7 DLLLILGTSLEVEP-FASLTEAVRSSVPRLLINRD------------------------LVGP--------------LAWHPR--SRDVAQLGDVVHG---------------------V 370 hSIRT4/Q9Y6E7 DSLLVVGSSLQVYSGYRFILTAWEKKLPIAILNIG-----------------------------------------------PTRSDDLACL---------------------------- 298 hSIRT5/Q9NXA8 DLCLVVGTSSVVYPAAMFAPQVAARGVPVAEFNTET--------------------------------------------TPATNRFRFHFQG--------------------------- 291 hSIRT6/Q8N6T7 DLSITLGTSLQIRPSGNLPLATKRRGGRLVIVNLQP--------------------------------------------TKHDRHADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLE 283 hSIRT7/Q9NRC8 DTILCLGSSLKVLKKYPRLWCMTKPPSRRPKLYIVN-----------------------------------------LQWTPKDDWAALKLHGKCDDVMRLLMAELG------------- 327 \* : :\*:\* : . . ySIR2/P06700 AMVAQKCGWTIPHKKWNDLKNKNFKCQ------------------EKDKGVYVVTSDEHPKTL--------------------------------------------------------- 562 hSIRT1/Q96EB6 VTLLDQAAKSNDDLDVSESKGCMEEKPQEVQTSRNVESIAEQMENPDLKNVGSSTGEKNERTSVAGTVRKCWPNRVAKEQISRRLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCG 672 hSIRT2/Q8IXJ6 LALAELLGWKKELEDLVRREHASIDAQSGAGVPNPSTSASPKKSPPPAKDEARTTEREKPQ----------------------------------------------------------- 389 hSIRT3/Q9NTG7 ESLVELLGWTEEMRDLVQRETGKLDG--------------PDK----------------------------------------------------------------------------- 399 hSIRT4/Q9Y6E7 -KLNSRCGELLPLIDPC------------------------------------------------------------------------------------------------------- 314 hSIRT5/Q9NXA8 -----PCGTTLPEALACHENETVS------------------------------------------------------------------------------------------------ 310 hSIRT6/Q8N6T7 RALPPLPRPPTPKLEPKEESPTRINGSIPAGPKQEPCAQHNGSEPASPKRERPTSPAPHRPPKRVKAKAVPS------------------------------------------------ 355 hSIRT7/Q9NRC8 LEIPAYSRWQDPIFSLATPLRAGEEGSHSRKSLCRSREEAPPGDRGAPLSSAPILGGWFGRGCTKRTKRKKVT----------------------------------------------- 400** 

Fig. 2. Alignment of centre core of human sirtuins family in comparison with yeast sir2 (previous page). The figure represent Clustalw alignment from indicated yeast and human sirtuins indicated by their UniProtKB accession numbers. In yeast sir2, the deacetylase sirtuin-type dominium is from 245 to 529 (red arrow) that correspond with the highest homology sequence among the members of the family. The NAD+ binding domains are indicated in green, there are the most conserved domains in the whole family. The active site is determined by a histidine at 364 position of sir2 that acts as a proton acceptor, the key histidines in other members have been determined in silico by homology. Although it has been indicated that these enzymes do not bind zinc, probable cystein residues able to bind zinc are also conserved in some of the members of the family (in blue). Regarding regulation, in sir2 two points of regulation by phosphorylation, phosphorylation at serine 23 and at tyrosine 400 have been determined (violet residues). None of them are conserved residues in human sirt forms. Further, in SIRT1, modifications at cysteines 395 and 398 by snitrosylation impede the binding of NAD+ and then, the activity of the enzyme.


Table 2. Pairwise comparison of the aminoacid sequences among yeast (*Saccharomyces cerevisiae*) SIR2 and higher homologues in model animals: fission yeast (Saccharomyces pombe); fly (*Drosophila melanogaster*); worm (*Caenorhabditis elegans*); zebrafish (*Dario rerio*); mice (*Mus musculus*) and human (*Homo sapiens*). The percentage of identity in comparison with *S. cerevisiae* sir2 protein is indicated. From BLAST analysis of the indicated proteins.

Among the other human sirtuins, SIRT4 and SIRT5 are mitochondrial sirtuins that show predominant ADP-ribosyl-transferase activity and a weak deacetylase activity and are involved in urea cycle regulation (Nakagawa & Guarente, 2009). On the other hand, SIRT6 and SIRT7 are considered as members of another subclass of sirtuins involved in reparation of DNA and the control of ribosomal RNA production through cell cycle (Lombard et al., 2008). Although it has been described that sirtuins does not bind zinc, Sir2, SIRT1, -2 and 3 share

four proximal cysteines that can indicate the possibility of binding zinc (figure 2). These four Cys are highly conserved among these sirtuins and just following the catalytic histidine. Then, although this Zinc ion must be not involved in the catalytic activity, their presence can be important for the maintenance of the structure of the sirtuin. In fact, recently Sanders and coworkers (Sanders et al., 2010) have shown that the four-cysteine metal binding site resembles the Zn-ribbon structure of transcription factors such as TF-IIS, TF-IIN and RNA polymerase II subunit RPB9. Further, although the Zinc-binding site is too far from catalytic domain, its presence is important for the activity of the enzyme since the change of any cystein to alanine or addition of zinc chelators inhibits the *in vitro* deacetylase activity of sirtuins (Min et al., 2001).

Regarding post-translational regulatory mechanisms, sirtuins can be regulated by phosphorylation and sumoylation. In fact, in SIRT1 thirteen residues have been found to be phosphorylated *in vivo* (Sasaki et al., 2008) indicating a high ratio of regulation by kinases. Further, dephosphorylation by protein phosphatases *in vitro* results in the decrease of the NAD+-dependent deacetylase activity in SIRT1. On the other hand, sumoylation of SIRT1 at

Sirtuin-Dependent Metabolic Control and Its Role in the Aging Process 101

al., 2010). On the other hand, the contrary effect of aging has been reported. In rats, an age-

Caloric restriction (CR) is the only dietary modification able to extend median and maximum lifespan in a number of organisms from yeast to mammals (Lomb et al., 2010). The effect of CR on lifespan extension is thought to be dependent on multiple different signaling pathways. CR decreases the activity of pro-aging pathways such as oxidative stress and insulin and growth hormone signaling whereas it stimulates the endogenous capacity of the cells against stress including antioxidant mechanisms (Qiu et al., 2010), DNA repair capacity and autophagy (Morselli et al., 2010). Further, the activity of mitochondria is modified in CR. Under CR, mitochondria show higher efficiency with lower reactive oxygen

Many of the effects of CR on longevity have been associated to the induction of sirtuin activity in cells (Cohen et al., 2004). Studies performed in mice have demonstrated that SIRT1 protein levels increases during CR in many tissues including brain, white adipose tissue, muscle, liver and kidney (Kanfi et al., 2008). Moreover, loss-of-function and gain-of-function mouse studies have provided genetic evidences that indicate that SIRT1 is a key factor in the physiological response to CR (Imai, 2009). It is also important to highlight that SIRT1 has been related to the central response to low nutritional availability at the hypothalamus level probably playing an important role in the regulation of the whole metabolism in mammals (Satoh et al., 2010). Further, SIRT6 levels are also modulated by nutrient availability in a p53-independent mechanism. SIRT6 modulation is mainly through the stabilization of protein levels but not via

The practice of exercise has been also considered to promote longevity and activate common pathways to CR probably by producing a metabolic stress in the organism (Lanza et al., 2008). Then, as in the case of CR, exercise also modulates the levels of sirtuins. In muscle, SIRT1 levels increases along aging and exercise training further increase the relative activity of this sirtuin (Koltai et al., 2010) indicated by an strong inverse correlation between nuclear activity of SIRT1 and the level of acetylated proteins. On the other hand, age-associated increase in SIRT6 levels is attenuated by exercise (Koltai et al., 2010). Exercise also increases SIRT3 expression in muscle and its activity is associated with a higher activity of AMP-dependent protein kinase (AMPK), cAMP-response element binding (CREB) and Peroxisome proliferatoractivated receptor gamma coactivator 1-α (PGC1α) indicating its importance in mitochondrial

biogenesis in muscle fibers especially in respiratory type I fibers (Palacios et al., 2009).

In heart, the aging-related decrease in MnSOD and catalase expression accompanied by the increase in oxidative damage levels indicated by TBARS and 4-HNE has been related to the decrease in the expression of SIRT1 (Ferrara et al., 2008). Exercise increases SIRT1 levels in heart reverting aging-related effect on MnSOD and catalase levels and its regulatory transcription factor, FOXO3a levels (Ferrara et al., 2008). Exercise not only modulates sirtuin levels in muscle or heart but also can modulate sirtuin levels and activity in central nervous system. Further, the modulation of SIRT1 by natural polyphenolic flavonoids such as resveratrol or quercetin may exert important beneficial effects in exercise performance

Sirtuin expression is also altered in cancer cells (Ashraf et al., 2006). This fact is important because these cells show a distinctive metabolism and higher growth in comparison with nontransformed cells. The different pattern of sirtuin expression in tumoral cells would confer to these cells higher resistance against exogenous agents and also control a different metabolism.

related increase in SIRT1 levels has been shown in skeletal muscle (Koltai et al., 2010).

species production (Lopez-Lluch et al., 2006).

increase of SIRT6-gene transcription (Kanfi et al., 2008).

(Lappalainen, 2011).

Lys734 residue has been also reported (Yang et al., 2007). Sumoylation consist in the binding of small ubiquitin-like modifier (SUMO) proteins to lysine residues (Hay, 2001). Binding of SUMO protein to SIRT1 increases its deacetylase activity and mutation of SIRT1 at the Lys734 residue or desumoylation by the nuclear desumoylase SENP1 reduces the activity (Yang et al., 2007).

Finally, another regulatory mechanism also establishes a relationship of sirtuins with metabolism. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys150 residue and binds to Siah1. Further, the complex moves to the nucleus since Siah1 show a nuclear localization signal. In the nucleus, Siah1 interacts with SIRT1 and other proteins. By this mechanism, S-nitrosylation of SIRT1 by GAPDH inhibits its deacetylase activity but specifically in the nucleus (Kornberg et al., 2010).

#### **2.1 Enzymatic activity of sirtuin**

As it has been above indicated (figure 1) sirtuins bind a Nε-acetyl-lysine of the target protein and deacetylate it by using NAD+ as substrate and producing NAM and 2'-O-AADPR (Hirsch & Zheng, 2011). However, this mechanism is no completely clear. In the case of SIRT6, although the deacetylation of histone 3 by SIRT6 has been described (Kawahara et al., 2009), other authors indicate that the main activity of this sirtuin is the ADP-ribosylation (Liszt et al., 2005). However, more recent studies indicate that the ADP-ribosyl-transferase of sirtuins could be only some inefficient side reactions of the deacetylase activity without any relevant physiological role (Du et al., 2009).

#### **2.2 Subcellular localization of sirtuins**

One of the key facts that determine the main targets of the different members of the sirtuin family is their respective subcellular localization. SIRT1 is found in the cell in both, the cytosol and the nucleus although it seems that nuclear localization is the most prevalent. However, recent research has demonstrated that SIRT1 is mainly sequestered in cytosol in highly glycolitic tumoral cells (Stunkel et al., 2007) indicating a metabolic-dependent localization of this deacetylase. On the other hand, SIRT3 is predominantly found in mitochondrial matrix (Schwer et al., 2002) although some studies have shown nuclear and also cytosolic locations (Sundaresan et al., 2008) whereas other authors have reported an exclusive mitochondrial localization (Cooper & Spelbrink, 2008). In the case of SIRT2, this sirtuin appears to be exclusively cytoplasmic (North & Verdin, 2007). SIRT4 and SIRT5 are located in the inner mitochondrial membrane or matrix (Michishita et al., 2005) and SIRT6 and SIRT7 are located in the nucleus (Schwer & Verdin, 2008).

#### **2.3 Modulation of sirtuin levels**

Acting as metabolic sensors, these proteins respond to many processes that affect the energetic balance in the organism including aging, dietary interventions, fasting or exercise. Aging progress is associated with a gradual decline of several physiological processes in the organism. In heart, age-related in SIRT1, decline is accompanied by a higher level of oxidative stress and the decrease in the expression of endogenous antioxidant enzymes and their regulators (Ferrara et al., 2008). In central nervous system, aging results in decreased activity of SIRT1 in cerebellum that leads to the increase in acetylation of protein residues specially affecting motor function (Marton et al., 2010). In cell culture models, cellular senescence induced by ionizing radiation is accompanied by the decrease in the levels of SIRT1 (Hong et

Lys734 residue has been also reported (Yang et al., 2007). Sumoylation consist in the binding of small ubiquitin-like modifier (SUMO) proteins to lysine residues (Hay, 2001). Binding of SUMO protein to SIRT1 increases its deacetylase activity and mutation of SIRT1 at the Lys734 residue or desumoylation by the nuclear desumoylase SENP1 reduces the activity

Finally, another regulatory mechanism also establishes a relationship of sirtuins with metabolism. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys150 residue and binds to Siah1. Further, the complex moves to the nucleus since Siah1 show a nuclear localization signal. In the nucleus, Siah1 interacts with SIRT1 and other proteins. By this mechanism, S-nitrosylation of SIRT1 by GAPDH inhibits

As it has been above indicated (figure 1) sirtuins bind a Nε-acetyl-lysine of the target protein and deacetylate it by using NAD+ as substrate and producing NAM and 2'-O-AADPR (Hirsch & Zheng, 2011). However, this mechanism is no completely clear. In the case of SIRT6, although the deacetylation of histone 3 by SIRT6 has been described (Kawahara et al., 2009), other authors indicate that the main activity of this sirtuin is the ADP-ribosylation (Liszt et al., 2005). However, more recent studies indicate that the ADP-ribosyl-transferase of sirtuins could be only some inefficient side reactions of the deacetylase activity without any

One of the key facts that determine the main targets of the different members of the sirtuin family is their respective subcellular localization. SIRT1 is found in the cell in both, the cytosol and the nucleus although it seems that nuclear localization is the most prevalent. However, recent research has demonstrated that SIRT1 is mainly sequestered in cytosol in highly glycolitic tumoral cells (Stunkel et al., 2007) indicating a metabolic-dependent localization of this deacetylase. On the other hand, SIRT3 is predominantly found in mitochondrial matrix (Schwer et al., 2002) although some studies have shown nuclear and also cytosolic locations (Sundaresan et al., 2008) whereas other authors have reported an exclusive mitochondrial localization (Cooper & Spelbrink, 2008). In the case of SIRT2, this sirtuin appears to be exclusively cytoplasmic (North & Verdin, 2007). SIRT4 and SIRT5 are located in the inner mitochondrial membrane or matrix (Michishita et al., 2005) and SIRT6

Acting as metabolic sensors, these proteins respond to many processes that affect the energetic balance in the organism including aging, dietary interventions, fasting or exercise. Aging progress is associated with a gradual decline of several physiological processes in the organism. In heart, age-related in SIRT1, decline is accompanied by a higher level of oxidative stress and the decrease in the expression of endogenous antioxidant enzymes and their regulators (Ferrara et al., 2008). In central nervous system, aging results in decreased activity of SIRT1 in cerebellum that leads to the increase in acetylation of protein residues specially affecting motor function (Marton et al., 2010). In cell culture models, cellular senescence induced by ionizing radiation is accompanied by the decrease in the levels of SIRT1 (Hong et

its deacetylase activity but specifically in the nucleus (Kornberg et al., 2010).

(Yang et al., 2007).

**2.1 Enzymatic activity of sirtuin** 

relevant physiological role (Du et al., 2009).

and SIRT7 are located in the nucleus (Schwer & Verdin, 2008).

**2.2 Subcellular localization of sirtuins** 

**2.3 Modulation of sirtuin levels** 

al., 2010). On the other hand, the contrary effect of aging has been reported. In rats, an agerelated increase in SIRT1 levels has been shown in skeletal muscle (Koltai et al., 2010).

Caloric restriction (CR) is the only dietary modification able to extend median and maximum lifespan in a number of organisms from yeast to mammals (Lomb et al., 2010). The effect of CR on lifespan extension is thought to be dependent on multiple different signaling pathways. CR decreases the activity of pro-aging pathways such as oxidative stress and insulin and growth hormone signaling whereas it stimulates the endogenous capacity of the cells against stress including antioxidant mechanisms (Qiu et al., 2010), DNA repair capacity and autophagy (Morselli et al., 2010). Further, the activity of mitochondria is modified in CR. Under CR, mitochondria show higher efficiency with lower reactive oxygen species production (Lopez-Lluch et al., 2006).

Many of the effects of CR on longevity have been associated to the induction of sirtuin activity in cells (Cohen et al., 2004). Studies performed in mice have demonstrated that SIRT1 protein levels increases during CR in many tissues including brain, white adipose tissue, muscle, liver and kidney (Kanfi et al., 2008). Moreover, loss-of-function and gain-of-function mouse studies have provided genetic evidences that indicate that SIRT1 is a key factor in the physiological response to CR (Imai, 2009). It is also important to highlight that SIRT1 has been related to the central response to low nutritional availability at the hypothalamus level probably playing an important role in the regulation of the whole metabolism in mammals (Satoh et al., 2010). Further, SIRT6 levels are also modulated by nutrient availability in a p53-independent mechanism. SIRT6 modulation is mainly through the stabilization of protein levels but not via increase of SIRT6-gene transcription (Kanfi et al., 2008).

The practice of exercise has been also considered to promote longevity and activate common pathways to CR probably by producing a metabolic stress in the organism (Lanza et al., 2008). Then, as in the case of CR, exercise also modulates the levels of sirtuins. In muscle, SIRT1 levels increases along aging and exercise training further increase the relative activity of this sirtuin (Koltai et al., 2010) indicated by an strong inverse correlation between nuclear activity of SIRT1 and the level of acetylated proteins. On the other hand, age-associated increase in SIRT6 levels is attenuated by exercise (Koltai et al., 2010). Exercise also increases SIRT3 expression in muscle and its activity is associated with a higher activity of AMP-dependent protein kinase (AMPK), cAMP-response element binding (CREB) and Peroxisome proliferatoractivated receptor gamma coactivator 1-α (PGC1α) indicating its importance in mitochondrial biogenesis in muscle fibers especially in respiratory type I fibers (Palacios et al., 2009).

In heart, the aging-related decrease in MnSOD and catalase expression accompanied by the increase in oxidative damage levels indicated by TBARS and 4-HNE has been related to the decrease in the expression of SIRT1 (Ferrara et al., 2008). Exercise increases SIRT1 levels in heart reverting aging-related effect on MnSOD and catalase levels and its regulatory transcription factor, FOXO3a levels (Ferrara et al., 2008). Exercise not only modulates sirtuin levels in muscle or heart but also can modulate sirtuin levels and activity in central nervous system. Further, the modulation of SIRT1 by natural polyphenolic flavonoids such as resveratrol or quercetin may exert important beneficial effects in exercise performance (Lappalainen, 2011).

Sirtuin expression is also altered in cancer cells (Ashraf et al., 2006). This fact is important because these cells show a distinctive metabolism and higher growth in comparison with nontransformed cells. The different pattern of sirtuin expression in tumoral cells would confer to these cells higher resistance against exogenous agents and also control a different metabolism.

Sirtuin-Dependent Metabolic Control and Its Role in the Aging Process 103

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

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

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

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),

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-

reactivation can produce beneficial effects (Tang, 2011).

activity (Kelly et al., 2009).

related to the activity of these enzymes.

SIRT1 seems to be a p53-dependent antiapoptotic factor.

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