Sirtuins form a family of NAD+-dependent deacetylases highly conserved from bacteria and lower eukaryotes to humans (Fyre 1999, 2000). Sir2 name originates from the gene involved in cellular regulation in yeast. Sirtuins affect energy metabolism and stress responses. Some sirtuins extend longevity in many organisms via their sensitivity towards NAD+. Mammalian sirtuins (SIRT1-7) play a vital role in aging, energy metabolism and cell survival in many tissues (Morris 2013). The sirtuins belong to the class III family of histone deacetylases and utilize NAD+ as a cofactor for their enzymatic activity, yielding deacetylated O-acetyl-ADP-ribose and nicotinamide (Landry et al. 2000). In addition to deacetylase activity, some sirtuins also have other enzymatic activities. Sirtuins are expressed in all metabolically active tissues.
2.2.1 Types of sirtuins
Sirtuins are divided into five classes based on their conserved amino acid core domain (Fyre, 2000). There are seven sirtuins in mammals (SIRT1-7), and all of them use NAD+ as a co-substrate (Figure 2). SIRT1, sirtuin 2 (SIRT2) and sirtuin 3 (SIRT3) are the mammalian sirtuins belonging to class I sirtuins. SIRT1 shares the highest sequence similarity with yeast Sir2 and Hst1, and SIRT2 and SIRT3 with Hst2 (North et al. 2003). Sirtuin 4 (SIRT4) belongs to class II, sirtuin 5 (SIRT5) to class III, and sirtuin 6 (SIRT6) and sirtuin 7 (SIRT7) to class V sirtuins.
Mammalian sirtuins have a conserved sirtuin core domain but have different functions.
SIRT1 is predominantly localized in the nucleus (Michishita et al. 2005), but it can also be found in the cytoplasm (Byles et al. 2010) and in some conditions in mitochondria (Aquilano et al.
2010). The localization of SIRT1 varies in different tissues under specific conditions depending on nutrient availability and sensing of the metabolic status. SIRT1 shuttles between the nucleus and cytoplasm, and this mechanism regulates its activity (Tanno et al. 2007). For example, calorie restriction (CR) influences the nuclear localization of SIRT1 (Shinmura et al. 2008). SIRT2 is primarily located in the cytoplasm but translocates to the nucleus under certain conditions (Vaquero et al. 2006). SIRT3, SIRT4 and SIRT5 are located in the mitochondria, and SIRT6 in the nucleus. SIRT7 is a nuclear protein specifically localized in the nucleolus (Vassilopoulus et al.
2011, Kupis et al. 2016).
Figure 2. Types of sirtuins, their primary structures and their subcellular localization. The figure shows mammalian sirtuins (SIRT1-7) in alignment with yeast Sir2. The conserved, catalytic domains of sirtuins are in yellow. Numbers represent the amino acids in the proteins. NLS- Nuclear localization sequence; MTS- Mitochondrial targeting sequence (Guarente 2013).
All sirtuins exhibit deacetylase activity. In this enzymatic reaction, sirtuins utilize NAD+ as a substrate to remove the acetyl groups from ε-acetyl lysine residues of target proteins. The lysine-bound acetyl group is transferred to the 2’-OH position of ADP-ribose, eventually generating a deacetylated protein and producing nicotinamide and 2’-O-acetyl-ADP-ribose (Cantó and Auwerx 2012) (Figure 3). SIRT2 also acts as demyristoylase (Teng et al. 2015), SIRT3 as decrotonylase (Tan et al. 2010), SIRT4 and SIRT6 act as mono-ADP-ribosyl transferases in reactions which involve the transfer of ADP-ribosyl moiety of NAD+ to a substrate protein.
SIRT4 acts as a lipoamidase and SIRT5 acts as demalonylase, desuccinylase and deglutarylase (Kupis et al. 2016). In addition to their deacetylase activity, mammalian sirtuins are also capable of catalyzing long-chain deacylation, demonstrated by the decrotonylase activity of SIRT1 and SIRT2 and delipoylation activity of SIRT1, SIRT2, SIRT3, and SIRT4 (Feldman et al. 2013).
Figure 3. The NAD+-dependent SIRT1 deacetylase reaction (Canto and Auwerx 2012).
2.2.2 Sir2, the prototypic sirtuin
The SIR2 gene was originally identified and characterized as one of the genes that regulates selected loci in Saccharomyces cerevisiae (Klar et al. 1979). Sir2 overexpression was initially demonstrated to promote histone deacetylation (Braunstein et al. 1993). The role of Sir2 in the regulation of transcriptional silencing at mating-type loci, telomeres and extrachromosomal rDNA circles suggested the involvement of Sir2 in extending the life span of yeast (Sinclair and Guarente 1997). The significant step in elevating Sir2 to the core of aging research was the discovery that overexpression of Sir2 alone can promote longevity in yeast (Kaeberlein et al.
1999). Imai et al. first studied the dependence of yeast and mouse Sir2 proteins on NAD+ for their histone deacetylase activity (Imai et al. 2000). Other studies showed the NAD+-dependent histone deacetylase activity of bacterial and human Sir2 proteins (Smith et al. 2000, Landry J et al. 2000). SIR2 gene is evolutionarily conserved from bacteria to humans (Brachmann et al. 1995).
The overexpression of Sir2 homologs in flies, nematodes and mice was found to increase the lifespan of the organisms (Tissenbaum and Guarente 2001, Rogina and Helfand 2004, Viswanathan and Guarente 2011; Banerjee et al. 2012). SIRT1 is the mammalian homolog of yeast Sir2 and the most extensively studied and characterized sirtuin.
2.2.3. The roles of sirtuins in type 2 diabetes
The numerous functions of sirtuins in glucose, lipid metabolism, energy homeostasis, insulin secretion and insulin sensitivity have projected them as promising therapeutic targets for treating T2D.
SIRT1 is well documented to mediate the beneficial effects of CR, and its role in metabolism and protection against T2D has been studied in detail. The role of SIRT1 in insulin signaling and nutrient sensing has been demonstrated in several animal models and in humans. SIRT1 enhances glucose-stimulated insulin secretion, regulates the insulin signaling pathway, protects
pancreatic β-cells from apoptosis and is vital for glucose metabolism (Kitada and Koya 2013).
SIRT1 enhances insulin sensitivity in metabolic tissues such as liver, skeletal muscle and adipose tissue by regulating several proteins and transcription factors through its deacetylase activity (Cao et al. 2016).
SIRT2 has been implicated in several metabolic functions, such as adipocyte differentiation through regulation of forkhead box protein O1 (FOXO1) (Jing et al. 2007) and fatty acid synthesis (Lin et al. 2013). It also promotes fatty acid oxidation and mitochondrial biogenesis via PGC-1α deacetylation in adipocytes with the inactivation of hypoxia-inducible factor 1α (HIF1α) (Krishnan et al. 2012).
SIRT3 is highly expressed in tissues rich in mitochondria such as brain, heart liver and BAT and functions as a mitochondrial deacetylase (Lombard et al. 2007). Increased triglycerides and low levels of FFA oxidation in the liver in the fasted state have been observed in SIRT3-deficient mice (Hirschey et al. 2010). SIRT3 regulates mitochondrial function, plays a crucial role in adaptive thermogenesis in BAT and is down-regulated in BAT of genetically obese mice.
Overexpression of SIRT3 enhances respiration and reduces membrane potential and production of reactive oxygen species (Shi et al. 2005). SIRT3 knock-out mice have increased oxidative stress and impaired insulin signaling in skeletal muscle (Jing E et al. 2011). Furthermore, SIRT3 protects high-fat-fed mice against IR and defects in skeletal muscle glucose uptake (Lantier et al.
2015).
SIRT4 is a mitochondrial sirtuin that mainly acts as an ADP-ribosyl transferase. SIRT4 interacts with glutamate dehydrogenase (GDH) and downregulates it in mitochondria of pancreatic β- cells. Loss of SIRT4 activates GDH, eventually enhancing insulin secretion stimulated by amino acids (Hagis et al. 2006). Overexpression of SIRT4 leads to a decrease in glucose-stimulated insulin secretion (Ahuja et al. 2007), in contrast to SIRT1.
SIRT5 is also a mitochondrial sirtuin with several enzymatic activities. SIRT5 has a potent lysine desuccinylase activity and its loss causes hyper-succinylation of mitochondrial proteins and impaired fatty acid β-oxidation (Rardin et al. 2013). SIRT5 regulates glycolysis via lysine malonylation; glycolysis is suppressed in the absence of SIRT5 due to increased malonylation of glycolytic enzymes (Nishida et al. 2015). In a study on adipose tissue of BMI-discordant monozygotic twins, SIRT5 expression was found to correlate positively with insulin sensitivity and negatively with inflammation (Jukarainen et al. 2016).
SIRT6 overexpression in mice protects against the accumulation of fat and impaired glucose tolerance induced by high fat diet and regulates lipid homeostasis by down-regulating key genes involved in lipid metabolism (Kanfi et al. 2010). SIRT6 deficiency is associated with severe hypoglycemia, enhanced insulin signaling, and increased basal and insulin-stimulated glucose uptake (Xiao et al. 2010). SIRT6 also functions as ADP-ribosyltransferase (Liszt et al. 2005), shows anti-inflammatory properties, and is downregulated in diabetic atherosclerotic plaques (Balestrieri et al. 2015). The deficiency of SIRT6 blocks adipocyte differentiation (Chen et al.
2017).
SIRT7 is a nuclear sirtuin that also regulates mitochondrial function. SIRT7 expression correlates positively with nuclear DNA encoded mitochondrial genes, and SIRT7 deficient mice have multi-systemic mitochondrial dysfunction (Ryu et al. 2014). SIRT7 has been found to promote obesity in mice (Cioffi et al. 2015), but in another study SIRT7 expression was decreased in obesity and increased after weight loss (Rappou et al. 2016).