Implications of altered sirtuins in metabolic regulation and oral cancer

SIRT1

SIRT1 is the most extensively investigated of the seven mammalian sirtuins (Brooks & Gu, 2009). SIRT1 exhibits the highest homology with yeast Sir2, which delays the ageing process and prolongs the lifespan in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster under caloric restriction (CR) (Burnett et al., 2011; Kaeberlein, McVey & Guarente, 1999; Chen & Guarente, 2007). The SIRT1 protein is largely found in the nucleus but can also be shuttled between the cytosol and nucleoplasm in various tissues in response to different environmental signals (Tanno et al., 2007) (Fig. 2). The SIRT1 protein contains 747 amino acids and has three independent domains: a central deacetylase domain (244–512 residues) that is conserved among species, a nuclear localization/export signal domain located in the N-terminal region (513–747 residues), and an essential activity domain located in the C-terminal region (1–180 residues). Specifically, the catalytic domain houses a substrate and an NAD⁺-binding pocket, and the N- and C-termini contain the regulatory and binding domains of the SIRT1 coactivator/corepressor, respectively. Additionally, SIRT1 contains an NLS (KRKKRK) at residues 41–46 and has thus been labelled a nuclear protein (Frye, 1999).

SIRT2

SIRT2 is the mammalian orthologue of yeast Hst2 (Perrod et al., 2001). Similar to yeast Sir2, Hst2 is upregulated by CR and oxidative stress and extends the lifespan through a Sir2-independent pathway (Lamming et al., 2005). SIRT2 is predominantly found in the cytoplasm (Fig. 2), where it acts principally as a deacetylase of microtubular proteins, such as alpha-tubulin (North et al., 2003). Thus, it serves as a regulator of the cell cycle, division, and proliferation (Li et al., 2007). Several lines of evidence suggest that SIRT2 has the ability to shuttle between the cytoplasm and the nucleus via cis-regulatory module (CRM)1-dependent active nuclear export (Inoue et al., 2007). The localization and function of SIRT2 are dependent on the differential splicing of its RNA (RNA splicing), which produces distinct isoforms with different N- and C-terminal extensions. Four different slice variants (also known as isoforms) are currently reported in the GenBank sequence database. However, only isoforms 1 and 2 have confirmed protein products with biological functionality. Isoform 1 is the full protein (389 aa), and isoform 2 lacks the first 37 aa from the N-terminus. Both isoforms 1 and 2 have a highly conserved catalytic core domain consisting of approximately 276 amino acid residues. A leucine-rich NES within the N-terminal region of these two isoforms has also been characterized (Pereira et al., 2018). Deletion of the NES leads to nucleocytoplasmic distribution, which suggests that the NES mediates cytosolic localization (North & Verdin, 2007).

SIRT3

SIRT3, which is a major NAD⁺-dependent protein deacetylase in mitochondria, plays an important role in regulating mitochondrial metabolism and energy production and is thought to be responsible for both the positive effects of exercise and caloric restriction on health (Schwer et al., 2002). Most studies support the notion that SIRT3 is localized in mitochondria (Onyango et al., 2002; Lombard et al., 2007; Verdin et al., 2010; Kratz et al., 2021), whereas other studies have suggested that SIRT3 might also be localized in the nucleus and cytoplasm (Scher, Vaquero & Reinberg, 2007; Sundaresan et al., 2008) (Fig. 2). Therefore, the localization and function of SIRT3 in various cellular compartments remain controversial. As a typical sirtuin, SIRT3 has a conserved enzymatic core (126–382 aa) responsible for its deacetylation function and acts in an NAD⁺-dependent manner. Two isoforms of SIRT3 are produced by alternative splicing in human cells.

The full-length 44-kDa form (isoform 1) contains 399 amino acid residues and is cleaved within mitochondria by matrix metalloprotease to a 28-kDa short form (which is denoted as isoform 2), which consists of an N-terminus missing 1–142 amino acid residues (Ota et al., 2004). The long isoform is found in mitochondria, the cytoplasm, and the nucleus, whereas the short isoform is found only in mitochondria (Scher, Vaquero & Reinberg, 2007).

SIRT4

In contrast to other sirtuins, SIRT4 has been relatively less extensively investigated. However, SIRT4 shares a conserved catalytic core of −270 amino acids. The core of SIRT4 has no C-terminal domain and contains a short, ∼44-aa N-terminal extension, which serves as a mitochondrial localization signal sequence (MLS) (Verdin et al., 2010; Kratz et al., 2021). The catalytic part of SIRT4 has a typical structure, which contains a large Rossmann fold domain and a small domain. The presence of an N-terminal MLS ensures that SIRT4 localizes within the mitochondrial matrix (Fig. 2). The cleavage of SIRT4 at amino acid 28 after its import into the mitochondria activates the enzymatic functions of the protein (Ahuja et al., 2007).

SIRT5

SIRT5, which is another mitochondrial sirtuin (mtSIRT), is the most recently investigated sirtuin. Similar to SIRT3 and SIRT4, SIRT5 is predominantly located in the mitochondrial matrix due to the presence of an N-terminal MTS (Verdin et al., 2010; Kratz et al., 2021). However, several studies have reported that SIRT5 is also found outside the mitochondria, although a fraction is observed in the cytosol (Matsushita et al., 2011) and peroxisomes (Chen et al., 2018), and very small amounts are also detected in the nucleus (Park et al., 2013) (Fig. 2). In humans, the SIRT5 gene encodes two major isoforms of the protein. SIRT5^iso1 is composed of 310 amino acids, whereas SIRT5^iso2 contains 299 amino acids and has a C-terminus that differs slightly from that of SIRT5^iso1 (Matsushita et al., 2011). More specifically, SIRT5^iso2 has 14 different residues (SHLISISSLIIIKN) between residues 286 and 299 and a missing aa in the 310th residue. Two additional human SIRT5 isoforms (SIRT5^iso3 and SIRT5^iso4) are also in the NCBI database (NR, 2018). The sequence of SIRT5^iso3resembles the sequence of SIRT5^iso1 with the exception of a lack of 18 internal amino acids (aa 189–206 are not present). SIRT5^iso4 is missing the first 108 amino acids of SIRT5^iso1, includes the MTS, and completely aligns with amino acids 109–310 of SIRT5^iso1. No data are currently available regarding the expression, localization, or functional properties of SIRT5^iso3 and SIRT5^iso4. A comparison of the structures of SIRT5 with those of other sirtuins reveals that the overall domain organization and folding of SIRT5 are similar to those of other reported sirtuin structures and that SIRT4 and SIRT5 exhibit many more overlapping regions (Soding, 2005).

SIRT6

SIRT6, a member of the sirtuin family of NAD⁺-dependent deacetylases, plays an important role in biological homeostasis, longevity, and various disease conditions. The SIRT6 structure consists of a large Rossmann fold and a smaller and structurally more varied sequence containing a zinc-binding domain. Unlike other sirtuins, SIRT6 does not contain a highly conserved cofactor-binding loop that aids in NAD⁺ binding but rather exhibits a helix structure that forms interactions with both ADP-ribose and 2′-N-acetyl-ADP-ribose (NAADPr) (Pan et al., 2011). The C-terminus is needed for proper nuclear localization, whereas the N-terminus is involved in the formation of chromatin associations and in enzymatic activity (Tennen, Berber & Chua, 2010). SIRT6 is reportedly a predominantly nuclear protein (Mostoslavsky et al., 2006) associated with telomeric heterochromatin regions (Michishita et al., 2008) (Fig. 2). The full-length isoform of SIRT6 (isoform 1, 39.1 kDa) contains 355 amino acids, and the shorter isoform (isoform 2, 36 kDa) lacks amino acids from the catalytic domain (amino acids 179–205) (Miteva & Cristea, 2014).

SIRT7

In humans, SIRT7 encodes a 400-amino-acid protein (in its full-length form) that functions as a class IV histone deacetylase that plays diverse roles in the ageing process, metabolic stress, and disease biology. Similar to other nuclear-localized sirtuins flanking the NLS (SIRT1 and SIRT6), SIRT7 is highly enriched in the nucleolus (Ford et al., 2006). A proportion of SIRT7 is also found close to the nuclear membrane in the cytoplasm (Kiran et al., 2013), which suggests that the shuttling of SIRT7 between various subcellular compartments is associated with and may be responsible for its multiple effects in diverse cellular responses (Tang et al., 2019; Zhang et al., 2016) (Fig. 2). Three protein-coding isoforms of SIRT7 are identified in the UniProtKB database due to alternative splicing mechanisms: (a) Q9NRC8-1, isoform 1, 400 aa, 44.9 kDa; (b) Q9NRC8-2, isoform 2, 183 aa, 20.4 kDa; and (c) Q9NRC8-3, isoform 3, 320 aa, 35.9 kDa. A search of the Ensembl Genome Browser revealed 21 splice variants in transcription products (ENSG00000187531), but there were only two protein-coding variants (SIRT7-210 and SIRT7-201 isoforms) (https://www.ensembl.org/index.html). To date, only a fragment of the SIRT7 N-terminus has been experimentally resolved, and the structure of the whole molecule remains to be determined (Priyanka et al., 2016). Based on a phylogenetic analysis, SIRT7 exhibits the highest degree of similarity to SIRT6 (Costantini et al., 2013).

SIRT1

SIRT1 is of central importance in regulating gluconeogenesis through its ability to deacetylate target proteins. SIRT1 can deacetylate CREB-regulated transcription coactivator 2 (CRTC2), which causes CRTC2 degradation and decreases hepatic glucose production (Liu et al., 2008). SIRT1 also enhances hepatic glucose output and gluconeogenesis through peroxisome proliferator-activated receptor (PPAR)g coactivator 1a (PGC-1α) and forkhead box O1 (FOXO1) (Houtkooper, Pirinen & Auwerx, 2012; Rodgers et al., 2005). PGC-1α is an important substrate of SIRT1 that plays a vital role in modulating glucose metabolism. Through PGC-1α, SIRT1 induces gluconeogenic genes in the liver. In contrast, in response to fasting and pyruvate, SIRT1 can modulate the PGC-1α-induced repression of glycolytic genes (Rodgers et al., 2005). Regarding glycolysis, SIRT1 can inhibit the process of glycolysis through the deacetylation and repression of glycolytic enzymes, such as phosphoglycerate mutase-1 (PGAM-1) (Hallows, Yu & Denu, 2012). SIRT1 reportedly suppresses glycolysis by repressing hypoxia-inducible factor 1α (HIF-1α) (Houtkooper, Pirinen & Auwerx, 2012; Lim et al., 2010). In pancreatic β cells, SIRT1 regulates insulin secretion by inhibiting the expression of UCP-2 and increasing ATP production to shut down the potassium channel, which allows the entry of calcium and the release of insulin (Bordone et al., 2006). In addition, SIRT1 promotes insulin expression by activating the expression of NeuroD and MafA (Zhou, Tang & Chen, 2018) SIRT1 and its activators reduce insulin resistance and diabetic complications and are thus potentially effective therapeutic targets for type 2 diabetes (T2D) (Ma et al., 2016; Kitada et al., 2019; Hegedus et al., 2020).

SIRT2

In terms of biochemical activities, SIRT2 is most similar to SIRT1, and its deacetylase activity can also promote glyconeogenesis. For example, SIRT2 can stabilize and deubiquitinate phosphoenolpyruvate carboxykinase (PEPCK-C), a rate-limiting enzyme in gluconeogenesis. During glucose deprivation, SIRT2 deacetylates PEPCK and increases gluconeogenesis (Jiang et al., 2011). SIRT2 maintains insulin sensitivity by acting as a glucose sensor. SIRT2 plays a vital role in supporting insulin resistance, and downregulation of SIRT2 improves insulin sensitivity (Lemos et al., 2017). A recent study also showed that SIRT2 ablation impairs glucose-stimulated insulin secretion by blocking glucokinase regulatory protein degradation and promoting aldolase A protein degradation, which causes a reduction in glycolytic flux (Zhou et al., 2021).

SIRT3

In addition to SIRT1, SIRT3 reportedly regulates glycolytic metabolism by maintaining the stability and regulating the activity of HIF-1α (Finley et al., 2011; Wang et al., 2020; Katwal et al., 2018). In contrast, a reduced level of SIRT3 is associated with high acetylation of peptidylprolyl isomerase D (cyclophilin D), which activates hexokinase II (HK2), a critical enzyme in glycolysis pathways (Wei et al., 2013). According to a recent report, the absence of SIRT3 is related to increases in the lysine 2-hydroxyisobutyrylation (Khib) levels of phosphofructokinase (PFK) and in glycolysis (Perico et al., 2021). SIRT3 can also initiate glucose synthesis by activating glutamate dehydrogenase (GDH) (Li et al., 2019; Fu et al., 2022), which converts glutamate toα-ketoglutarate in mitochondria (Schlicker et al., 2008; Zou et al., 2017).

SIRT4

Unlike SIRT1-3, SIRT4 does not display NAD-dependent deacetylase activity and can regulate insulin secretion by using NAD for the ADP-ribosylation of GDH in pancreatic β cells (Haigis et al., 2006). SIRT4 downregulates the enzymatic activity of GDH and hinders the production of ATP from glutamate and glutamine to further promote insulin secretion (Haigis et al., 2006). In addition to GDH, SIRT4 is thought to regulate insulin secretion via various targets, including ADP/ATP carriers and the insulin-degrading enzymes ANT2 and ANT3 (Ahuja et al., 2007). Furthermore, SIRT4 can inhibit insulin secretion by promoting leucine catabolism (Wang & Wei, 2020).

SIRT5

SIRT5 possesses deacetylase- and NAD⁺-dependent demalonylase and desuccinylase activities. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme. In glycolysis, SIRT5 can regulate the activity of GAPDH by demalonylating its homodimerization interface residue, K184 (Nishida et al., 2015). The findings of a recent study suggest that SIRT5 may be positively correlated with insulin sensitivity (Jukarainen et al., 2016). Although SIRT5 plays multiple roles in the regulation of cellular metabolism, further research is needed to identify its direct substrates and determine its exact function.

SIRT6

SIRT6 is essential for the maintenance of glucose homeostasis. Similar to SIRT1, SIRT6 suppresses glycolysis by acting as a corepressor for HIF-1α (Zhong et al., 2010; Zeng et al., 2021). In glyconeogenesis, SIRT6 binds to and promotes the activity of 5GCN5 (general control nonrepressed protein), which acetylates PGC-1α. Acetylated PGC-1α activates PPAR γ to inhibit glyconeogenesis-related enzymes, such as PEPCK-C and G6P, thereby resulting in the inhibition of hepatic glucose production by repressing gluconeogenesis (Kugel & Mostoslavsky, 2014). SIRT6 also maintains glucose homeostasis by downregulating multiple members of the insulin signalling pathway, such as AKT, insulin receptor, and the insulin receptor substrates IRS1 IRS2, glucose transporter-1 (GLUT1), and glucose transporter-4 (GLUT4) (Xiao et al., 2010; Parenti et al., 2014; Liu et al., 2018; Huang et al., 2019; Yang et al., 2020; Wu et al., 2021; Tang & Fan, 2019).

SIRT7

SIRT7 also interacts with hypoxia-inducible factors. The overexpression of SIRT7 can reduce the protein levels of both HIF-1α and HIF-2α independently of its deacetylase activity (Hubbi et al., 2013; Wu et al., 2018). Moreover, mice lacking SIRT7 display better resistance to glucose intolerance and increased insulin sensitivity when fed fat-containing diets, which suggests that SIRT7 plays a crucial role in glucose metabolism (Yoshizawa et al., 2014).

SIRT1

SIRT1 can regulate lipid metabolism via its deacetylase activity. For instance, SIRT1 deacetylates and destabilizes sterol regulatory element-binding protein (SREBP)-1 and (SREBP)-2, which are transcription factors related to lipid metabolism, and thereby represses lipid synthesis and fat storage during fasting (Thiel, Guethlein & Rossler, 2021). In white adipose tissue, SIRT1 mediates corresponding effects on fat accumulation. SIRT1 binds to and functionally inhibits the fat regulator peroxisome proliferator-activated receptor- γ (PPARγ) by interacting with the PPARγ cofactor nuclear receptor corepressor (NCoR) and silencing the mediator of retinoid and thyroid hormone receptors (SMRT) (Zhou, Tang & Chen, 2018; Picard et al., 2004). A SIRT1/PPARγ/NCoR complex binds to conspecific DNA sites in PPAR- γ target gene promoter sequences and suppresses their transcription (Picard et al., 2004). Thus, genes involved in fatty acid accumulation and lipolysis can thus be negatively affected. SIRT1 also regulates hepatic lipid homeostasis by interacting with peroxisome proliferator-activated receptor -α (PPARα), a nuclear receptor for lipid homeostasis (Bougarne et al., 2018). PGC-1α is a direct substrate of PPARα (Vega, Huss & Kelly, 2000; Cheng, Ku & Lin, 2018), and SIRT1 alters PPARα signalling by deacetylating and activating the PPARα coactivator PGC-1α (Purushotham et al., 2009; Kalliora et al., 2019; Kosgei et al., 2020; Li et al., 2021). The loss of SIRT1 reduces PPARα signalling and impairs fatty acid β-oxidation (Purushotham et al., 2009). A growing body of evidence suggests that SIRT1 could be an important therapeutic target in preventing lipid metabolic diseases.

SIRT2

SIRT2 exerts a negative regulatory effect on adipogenesis through its deacetylase activity. By deacetylating FOXO1, SIRT2 suppresses adipogenesis in part through binding FOXO1 to PPAR γ and repressing its transcriptional activity (Wang & Tong, 2009). A recent study revealed that SIRT2 inhibits lipid accumulation partially by binding to and deacetylating the hepatocyte nuclear factor 4α (HNF4α) protein on lysine 458 to increase HNF4α stability (Ren et al., 2021). SIRT2 may be a promising target in the treatment of lipid metabolic disorders.

SIRT3

SIRT3 plays an essential role in the metabolic process of fatty acid oxidation (FAO). SIRT3 can deacetylate and reduce the enzymatic activity of long-chain acylCoA dehydrogenase (LCAD), a protein involved in FAO, during prolonged fasting to enhance FAO (Hirschey et al., 2010). According to a recent study, SIRT3 also regulates FAO by deacetylating liver kinase B1 (LKB1) and activating AMP-activated protein kinase (AMPK) (Li et al., 2020). Additionally, SIRT3 contributes to the prevention of nonalcoholic fatty liver disease. SIRT3 ameliorates lipotoxicity in hepatocytes by reducing the expression of stearoyl-CoA desaturase 1 (SCD1), a key lipogenic enzyme, to suppress lipogenesis (Zhang et al., 2020). SIRT3 can also deacetylate and stimulate the activity of 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) in the liver (Hirschey et al., 2011), which results in increased ketogenesis (Shimazu et al., 2010).

SIRT4

In contrast to SIRT3, SIRT4 negatively regulates FAO and stimulates lipogenesis by directly binding to, deacetylating, and repressing malonyl-CoA decarboxylase (MCD), an enzyme that produces acetyl-CoA from malonyl-CoA (Laurent et al., 2013b). In addition, by dampening the activity of SIRT1 and PPARα, SIRT4 can lead to a reduction in FAO in the liver (Laurent et al., 2013a). A recent study showed that SIRT4 positively functions as a regulator of branched-chain amino acid (BCAA) catabolism, promotes the expression of PPARγ in early adipogenesis, and consequently stimulates adipogenesis (Zaganjor et al., 2021).

SIRT5

The hepatic overexpression of SIRT5 can improve mitochondrial FAO in hepatocytes by desuccinylation proteins (Du et al., 2018). SIRT5-knockout mice also exhibit reduced FAO (Rardin et al., 2013). In parallel, SIRT5 can protect against acute kidney injury by regulating proximal tubule FAO (Chiba et al., 2019). The effect of SIRT5 on FAO may have potential therapeutic implications in the treatment of acute kidney injury. In addition, SIRT5 deficiency reduces the intracellular levels of α-ketoglutarate, and this reduction leads to higher levels of methylation at the promoters of the PPARγ and Prdm16 genes, which can repress brown adipogenesis (Seale, Kajimura & Spiegelman, 2009). According to a recent study, a SIRT5 inhibitor stimulates brown adipogenesis (Molinari et al., 2021).

SIRT6

SIRT6 plays a crucial role in lipid mobilization. By activating the adenosine monophosphate-activated protein kinase alpha (AMPK α) pathway, SIRT6 inhibits preadipocyte differentiation and lipid synthesis and works in concert with SIRT5 to decrease lipid deposition and inhibit cell cycle arrest of preadipocytes (Hong et al., 2020). Additionally, SIRT6 deficiency causes increases in triglyceride (TG) synthesis and long-chain fatty acid uptake and decreases fatty acid β-oxidation genes. Furthermore, the knockout of SIRT6 results in fatty acid liver disease due to TG accumulation (Kim et al., 2010). Under high-fat diets, SIRT6 maintains lipid homeostasis by downregulating genes specifically regulated by PPARγ (Kanfi et al., 2010). Furthermore, SIRT6 can bind to the DNA-binding domain of PPARγ, and this binding regulates its activity at promoters and consequently controls the expression of fatty acid transporters (Khan et al., 2021).

SIRT7

SIRT7 can interact with SIRT1 during adipogenesis. By inhibiting the activity of SIRT1, SIRT7 contributes to efficient adipocyte differentiation and thereby indirectly and efficiently activates PPARγ (Fang et al., 2017). Furthermore, SIRT7 can bind to and directly deacetylate PPARγ2 to regulate adipocyte lipogenesis (Akter et al., 2021). However, according to a previous study, SIRT7-knockout mice show liver steatosis as a result of suppressed ER stress (Shin et al., 2013). In light of this finding, further research on the mechanisms underlying the regulation of lipids by SIRT7 is needed.