Function of the SIRT3 mitochondrial deacetylase in cellular physiology, cancer, and neurodegenerative disease.

In mammals, seven members of the sirtuin protein family known as class III histone deacetylase have been identified for their characteristic features. These distinguished characteristics include the tissues where they are distributed or located, enzymatic activities, molecular functions, and involvement in diseases. Among the sirtuin members, SIRT3 has received much attention for its role in cancer genetics, aging, neurodegenerative disease, and stress resistance. SIRT3 controls energy demand during stress conditions such as fasting and exercise as well as metabolism through the deacetylation and acetylation of mitochondrial enzymes. SIRT3 is well known for its ability to eliminate reactive oxygen species and to prevent the development of cancerous cells or apoptosis. This review article provides a comprehensive review on numerous (noteworthy) molecular functions of SIRT3 and its effect on cancer cells and various diseases including Huntington's disease, amyotrophic lateral sclerosis, and Alzheimer's disease.

Introduction

SIRT3 is a NAD+‐dependent protein deacetylase that is a member of the silent information regulator 2 (SIR2) family (Giralt and Villarroya, 2012). SIRT3 can exert controls on a wide range of important biological activities including regulation of nuclear gene expression, metabolic control (Shi et al., 2005), neuroprotection (Kong et al., 2010), cardiovascular disease, cancer (Alhazzazi et al., 2011b), and aging (Bellizzi et al., 2005). SIRT3 is the only sirtuin protein reported to affect human lifespan (Kong et al., 2010; Brown et al., 2013; Kincaid & Bossy‐Wetzel, 2013). As it is localized exclusively in mitochondria, SIRT3 can regulate characteristic mitochondrial processes like protein deacetylation (Lombard et al., 2007). Some other organs and active metabolic tissues, including brown adipose tissue (BAT), heart, and kidney, were also reported to accommodate significant concentrations of SIRT3 (Palacios et al., 2009).

Among the mitochondrial sirtuins, only SIRT3 possesses robust NAD+‐dependent deacetylase activity (Lombard et al., 2007). SIRT3 can undergo proteolytic cleavage upon translocation to the mitochondrion. Full‐length (FL) SIRT3 is proposed to be inactive until it is translocated to and proteolytically processed within the mitochondrion to an active 28 kDa protein (Iwahara et al., 2012). Cleavage of SIRT3 at the mitochondrial localization sequence is known to activate deacetylase activity (Smith et al., 2008).

SIRT3 influences energy metabolism processes (e.g., tricarboxylic acid cycle, respiratory chain, fatty acid β‐oxidation, and ketogenesis) by targeting the responsible enzymes (Giralt and Villarroya, 2012). It also controls the flow of mitochondrial oxidative pathways and, ultimately, the rate of reactive oxygen species (ROS) production. SIRT3‐mediated deacetylation activates enzymes responsible for reducing ROS in protective action against oxidative stress‐dependent phenomena or diseases such as cardiac hypertrophy, aging, cancer, cardiac dysfunction, and neural degeneration. SIRT3 also plays a role in multiple additional metabolic processes from acetate metabolism to BAT thermogenesis, often by controlling mitochondrial pathways through the deacetylation of target enzymes (Shi et al., 2005).

The role of SIRT3 in physiology is crucial and an interesting subject for research. Protein acetylation regulates global mitochondrial function (Kim et al., 2006). Research implementing SIRT3‐knockout (KO) mice revealed many significant aspects of SIRT3 in physiology (Ahn et al., 2008). SIRT3 interacts with FOXO3a to activate antioxidant genes like manganese superoxide dismutase (MnSOD) and catalase, whose gene products reduce ROS while positively affecting disorders like cardiac hypertrophy and interstitial fibrosis (Sundaresan et al., 2009). SIRT3‐knockout mice are prone to age‐related disorders like cancer, cardiac hypertrophy, and metabolic syndrome (Choudhury et al., 2011; Hirschey et al., 2011). Frequent opening of mitochondrial permeability transition pore (mPTP) can lead to mitochondrial dysfunction. Note that mPTP is regulated by cyclophilin D (CypD) which is deacetylated by SIRT3. The absence of SIRT3 in cardiac muscle becomes a stimulus to increase the opening of mPTP. Thus, SIRT3 KO mice was found to suffer from hampering of mitochondrial function in heart with the sign of aging (Hafner et al., 2010). SIRT3‐knockout rodents also develop pulmonary arterial hypertension (Paulin et al., 2014). SIRT3 regulates stress response in hematopoietic stem cells (HSC) and improves regenerative capacity in aged HSCs, while it controls tissue homeostasis as well (Brown et al., 2013). SIRT3 can also manage NAD+ levels to regulate mitochondrial function, offering protection against liver injury associated with fatty liver (Kendrick et al., 2011) and/or acute kidney injury (Morigi et al., 2015). Because SIRT3 KO mice cannot maintain SOD2 homoeostasis and ROS level, they suffer from mild endothelial dysfunction when fed with high cholesterol diet (Winnik et al., 2016).

This study aims to provide a comprehensive review of the most relevant biological and pathophysiological functions of SIRT3 such as cancer control, neuroprotection, DNA repair, enhanced longevity, energy homoeostasis, oxidative stress tolerance, and many other mechanisms. To this end, most important studies conducted in this field were compiled and reviewed with respect to essential functions of SIRT3.

SIRT3 plays an important role in numerous molecular functions by controlling many crucial biological activities. As illustrated in Fig. 1, SIRT3 helps maintain the physiology of the body through various routes. (Refer to Table 1 for details.)

Figure 1

Molecular functions of SIRT3.

Table 1

Molecular functions of SIRT3

Molecular Function	Associated Enzyme/Protein	Activity of SIRT3	Location	Reference
Energy homeostasis	Absence of deacetylase	Reduction of ATP	Heart, kidney, liver	(Ahn et al., 2008)
	Protein acetylation	Increase in ATP	Mitochondria
Suppression of ROS	Increase of superoxide dismutase 2	Stimulates SIRT3 transcription, leads to SOD2 deacetylation and activation of oxidative stress	Mitochondria	(Chen et al., 2011)
	Binds to peroxisome proliferator‐activated receptor co‐activator‐1α	Stimulates SIRT3 to regulate adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis, and respiration	Muscle cells and hepatocytes	(Lin et al., 2005; Finck & Kelly, 2006)
Apoptosis	SIRT3 along with SIRT4	Inhibits apoptosis by decreasing NAD+ levels	Somatic cells	(Yang et al., 2007)
	Mitochondrial ribosomal protein L10	Regulates mitochondrial protein synthesis by deacetylation of ribosomal protein MRPL10	Mitochondria	(Yang et al., 2010)
Tumor suppression	Decrease of superoxide dismutase	Depletion of SIRT3 leads to tumor suppression	Mitochondria	(Chen et al., 2011)
	Decrease of superoxide dismutase	Loss of SIRT3 promotes transformation through an increase in chromosomal instability via increased production of ROS and altered intracellular metabolism	Mitochondria	(Kim et al., 2010)
	Human 8‐oxoguanine‐DNA glycosylase 1	SIRT3 prevents the degradation of protein and repairs mitochondrial DNA damage	Mitochondria	(Cheng et al., 2013)
Muscles	Binds to Ku70 in cardiac muscles	Physically binds and deacetylates Ku70 and promotes interaction of Ku70 with the pro‐apoptotic protein Bax	Cardiomyocytes	(Sundaresan et al., 2008)
	CREB phosphorylation in skeletal muscles	Exercise increases SIRT3 expression	Skeletal muscle cells	(Palacios et al., 2009)
	Phospho‐activation of AMPK in skeletal muscles	SIRT3 expression increases with fasting and caloric restriction and decreases with high‐fat diet. A caloric restriction regimen also leads to phospho‐activation of AMPK in muscle	Skeletal muscle cells	(Palacios et al., 2009)
Neurons	LKB1	SIRT3 deacetylates and activates LKB1, augmenting LKB1–AMPK pathway activity, thus protecting against an increase in NAD	Mitochondria	(Miao & St Clair, 2009)
	Activates MnSOD and catalase	Decreases cellular levels of ROS	Mitochondria	(Kawamura et al., 2010)
	Poly (ADP‐ribose) polymerase‐1 activation or protein transfection of NADase	NAD depletion leads to over‐expression of SIRT3, which induces ROS generation, thereby preventing neuronal death	Mitochondria	(Kim et al., 2011)
Caloric restriction	Increase in the deacetylase	Induces the expression of genes involved in mitochondrial biogenesis	Adipocytes	(Shi et al., 2005)
Diabetes	Increase mitochondrial enzyme acetyl‐CoA synthetase 2	Increases SIRT3 under ketogenic conditions	Intermembrane space of the mitochondrion	(Schwer et al., 2006)
Metabolism	Activation of acetyl‐CoA synthetase 2 and glutamate dehydrogenase	Deacetylates and activates the enzymes to enhance the Krebs cycle and oxidative phosphorylation	Mitochondria	(Hallows et al., 2006; Schlicker et al., 2008)
	Succinate dehydrogenase complex	NAD+‐dependent deacetylase, SIRT3 regulates the activity of enzymes	Mitochondria	(Cimen, Han et al. 2009)
Thermogenesis	Increase expression of protein PGC‐1α, uncoupling protein (UCP1), and a series of mitochondria‐related genes in the presence of both ADP and ribosyltransferase	Deacetylates SIRT3	Brown adipose tissue and mitochondrial inner membrane	(Shi et al., 2005)
Fatty acid oxidation	Increase in long‐chain acyl‐CoA dehydrogenase	Upregulation of SIRT3	Fasting in liver and brown adipose tissues	(Allison & Milner, 2007)

Significance of SIRT3 in metabolism

SIRT3 is characterized as a mitochondrial sirtuin protein that is localized in the mitochondrial matrix. It regulates the activity of several metabolic enzymes and Complex I subunit NDUFA9 by deacetylation (Ahn et al., 2008). Protein synthesis by oxidative phosphorylation (OXPHOS) is encoded by the mitochondrial genome, which is carried out by mitochondrial ribosomal protein L10 (MRP L10). However, MRP L10 generally remains in the acetylated form in the mitochondrial ribosome and becomes deacetylated by SIRT3 (Yang et al., 2010). SIRT3 deacetylates and activates acetyl‐CoA synthetase 2 and glutamate dehydrogenase (GDH), leading to enhancement of the Krebs cycle (Hallows et al., 2006; Schlicker et al., 2008). SIRT3 also regulates OXPHOS by activating acetyl‐CoA synthetase 2 and GDH (Ahn et al., 2008).

Keratinocyte differentiation is known to influence the formation/maintenance of the protective skin barrier (Bause et al., 2013; Chen et al., 2014). However, dysregulation in the balance of ROS homeostasis is known to impact keratinocyte differentiation. Loss of SIRT3 expression in keratinocytes increases superoxide levels and promotes the expression of differentiation markers, while overexpression decreases superoxide levels and reduces the expression of differentiation markers. However, it remains unclear how mitochondrial oxidative stress signaling induces and controls keratinocyte differentiation (Bause et al., 2013). Previous studies have shown that ROS signaling induces keratinocyte differentiation through the protein kinase C/activator protein‐1 (PKC/AP‐1) pathway (Rutberg et al., 1996; Bose et al., 2012). SIRT3 gene expression may in turn also be regulated through AP‐1 transcription factors as a possible regulatory pathway (Bellizzi et al., 2009). Cumulatively, SIRT3 is one of the most remarkable regulators in metabolism. It should be noted that the molecular control of SIRT3 itself is quite abstruse. The maintenance of mitochondrial DNA (mtDNA) is regulated by several components such as TFB1M and TFB2M which are known as very crucial factors determining the mitochondrial transcription specificity in human (Falkenberg et al., 2002). They are also regulated by nuclear respiratory factors (NRF1 and NRF2) (Gleyzer et al., 2005). For instance, as the role of NRF2 in the expression of SIRT3 promoter is significant, such mechanism can make substantial contribution to the mitochondrial biogenesis (Satterstrom et al., 2015).

SIRT3 in the protection of neurons

One of the important functions of SIRT3 is the protection of neurons attained by the interaction with NAD+ and poly (ADP‐ribose) polymerase‐1 (PARP‐1). PARP‐1 is a DNA repair nuclear enzyme that helps prevent chromatid exchange (Schreiber et al., 2006). However, some stressful conditions like excitotoxicity, ischemia, inflammation, and oxidative stress cause over‐activation of PARP‐1, which is the key pathway responsible for neuronal cell death in the aforementioned consequences (Virág & Szabó, 2002). Nevertheless, over‐activation of PARP‐1 selectively decreases cytosolic NAD+ (Alano et al., 2004, 2010); PARP‐1‐mediated NAD+ depletion then accompanies colossal ROS production in the neuronal cell, which increases the expression of mitochondrial SIRT3. An overexpression of SIRT3 suppresses ROS generation, thereby preventing neuronal death (Kim et al., 2011). SIRT3 can also directly mediate adaptive neuronal responses to bioenergetic, oxidative, and excitatory stress. According to some experiments made with SIRT3 KO mice, cortical neurons lacking SIRT3 exhibited sensitivity to above‐mentioned physiological challenges. On the contrary, SIRT3 gene delivery can restore resistance to stress. The absence of SIRT3 was thus accompanied by hyperacetylation of SOD2 and CypD (Cheng et al., 2016).

Amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disease, is characterized by the degeneration of both upper and lower motor neurons (Boillée et al., 2006; Zhao et al., 2013). Mutations in the related gene encoding Cu/Zn superoxide dismutase (SOD1) can affect an inherited form of ALS. Note that more than 150 types of SOD1 gene mutations have been described, while most of them are transmitted in an autosomal dominant pattern (Battistini et al., 2005). The exact mechanism underlying SIRT3‐mediated protection against mutant SOD1‐induced toxicity remains elusive; however, the co‐presence of SIRT3 and peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) protects against mitochondrial fragmentation and neuronal cell death (Kong et al., 2010; Kincaid & Bossy‐Wetzel, 2013). The mitochondrial SIRT3 promoter region carries an estrogen‐related receptor (ERR)‐binding element (ERRE) located 399‐ to 407‐bp downstream (Kong et al., 2010). As oxidative stress increases, PGC‐1α recruits ERRα, a well‐known mitochondrial regulator, to the ERRE. The overexpression of SIRT3 is thus executed based on this mechanism (Kong et al., 2010). SIRT3 lowers ROS levels by deacetylating SOD2, while increasing its enzymatic activity (Bruijn et al., 2004; Qiu et al., 2010; Someya et al., 2010). Furthermore, SIRT1 is known to upregulate SIRT3 transcription through PGC‐1α (Tao et al., 2010; Giralt et al., 2011) (Fig. 2). PGC‐1α also co‐activates NRF2, another novel regulator of SIRT3 (Satterstrom et al., 2015). As neuroprotection by SIRT3 in above disease is mediated through PGC1α, it may help find future remedies of such disorders.

Figure 2

SIRT3 protect neurons during cellular stress.

Function of SIRT3 in different muscle types

The expression of SIRT3 also plays a role in heart and skeletal muscles (Shi et al., 2005; Lombard et al., 2007). SIRT3 is a stress‐responsive deacetylase in cardiomyocytes and protects cells from genotoxic and oxidative stress‐mediated damage. As SIRT3 is a stress‐responsive deacetylase, its increased expression protects genotoxic and oxidative stress‐mediated cell death in murine cardiomyocytes. Under stressful conditions, SIRT3 levels increase in mitochondria as well as in cardiomyocyte nuclei. SIRT3 can then be physically bound to Ku70 for its deacetylation to promote interaction between Ku70 and the pro‐apoptotic protein Bax (Sundaresan et al., 2008). In addition, the antihypertrophic effects of exogenous NAD+ are also mediated through the activation of SIRT3 instead of SIRT1. As SIRT3 is able to deacetylate and activate liver kinase B1 (LKB1), it can augment LKB1–AMPK pathway activity. NAD+, while acting as an inhibitor of cardiac hypertrophic signaling, is regulated to protect against cardiac hypertrophy and heart failure (Miao & St Clair, 2009; Pillai et al., 2010). In mice, SIRT3 is activated by the natural biphenolic compound honokiol and protects cardiac muscles from hypertrophy (Fig. 3) (Sundaresan et al. 2009; Pillai et al., 2015).

Figure 3

SIRT3‐mediated protection of cardiac and skeletal muscle during hypertrophy and fasting, respectively.

In skeletal muscles, SIRT3 responds to both exercise and CREB phosphorylation with PGC‐1α upregulation of nutritional signals to coordinate downstream molecular responses (Palacios et al., 2009; Hokari et al., 2010) (Fig. 3). In skeletal muscle, SIRT3 protein level is also sensitive to diet as its expression increases by fasting and caloric restriction and decreases after a high‐fat diet. A caloric restriction regimen also leads to the phospho‐activation of AMPK in muscle (Palacios et al., 2009). When skeletal muscles were treated with near‐infrared light, an increase in oxidative stress was found with increases in the levels of upstream mitochondrial regulatory AMPK, p38, PGC‐1α, and SIRT1 proteins along with reduced levels of RIP140; however, mitochondrial regulation/content remained unaltered for SIRT3, Tfam, NRF‐1, cytochrome c, and ETC subunits (Nguyen et al., 2014). Consequently, the mechanism underlying such signaling in muscle cells remains unclear.

SIRT3 in DNA repair

SIRT3 is capable of affecting mitochondrial OXPHOS by regulating the function of mitochondrial enzymes (Haigis et al., 2012). Human mitochondrial DNA (mtDNA) contains a number of genes that encode 13 polypeptides, two rRNAs, and 22 tRNA molecules; the 13 polypeptides are known to influence mitochondrial respiration and OXPHOS (Schon, 2000). Mutation of the mitochondrial genome may cause some inherited diseases; remarkable progress has been made from researches in mtDNA mutations with respect to aging and cancer (Taylor & Turnbull, 2005). Moreover, mtDNA is considerably more prone to oxidative damage than the nuclear genome (Richter et al., 1988). Oxygen‐free radicals generated by mitochondrial respiration or by exposure to ionizing radiation (or chemicals) are responsible for damaging DNA. Oxidative damage can thus change purine and pyrimidine bases in DNA to 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) (Grollman & Moriya, 1993); however, human 8‐oxoguanine‐DNA glycosylase 1 (OGG1) is a newly identified target protein of SIRT3 that functions in DNA repair by expunging 8‐oxoG from damaged genomic DNA (Cheng et al., 2013). SIRT3 becomes physically associated with OGG1 to prevent degradation of the OGG1 protein and controls its incision activity when DNA glycosylase is acetylated. Furthermore, SIRT3 plays a critical role in repairing mitochondrial DNA (mtDNA) damage, protecting mitochondrial integrity, and preventing apoptotic cell death under oxidative stress by regulating the acetylation and turnover of OGG1 (Cheng et al., 2013).

SIRT3 in aging

One of the most interesting attributes of SIRT3 is its potential to promote the extension of lifespan (Rose et al., 2003; Halaschek‐Wiener et al., 2009). A VNTR polymorphism prevails in the SIRT3 gene as a potential regulator of SIRT3 expression. In light of the high abundance of SIRT3 in long‐lived individuals, a potential link was suggested between SIRT3 and longevity (Bellizzi et al., 2005). A better understanding on SIRT3‐dependence of expanded lifespan will indeed help broaden the spectrum of research in this field. The FOXO1, FOXO3a, FOXO4, and FOXO6 genes encode the FOXO family of transcription factors and are human homologs of the daf‐16 gene in Caenorhabditis elegans, the product of which contributes to the regulation of nematode lifespan (Shi et al., 2005). FOXO transcription factors appear to regulate a wide variety of intracellular processes such as cellular resistance to oxidative stress and general metabolism (Burgering & Kops, 2002; Accili & Arden, 2004). It is also acknowledged that nuclear sirtuins interact with the activity of FOXO family proteins under specific cellular conditions, as FOXO3a is known to physically interact with SIRT3 in mitochondria (Fig. 4) (Wang et al., 2007; Guarente, 2008). Human colon carcinoma (HCT116) cells were used to overexpress wild‐type and/or a catalytically inactive dominant species of negative SIRT3 (Jacobs et al., 2008). Overexpression of the wild‐type SIRT3 gene increases FOXO3a DNA‐binding activity and FOXO3a‐dependent gene expression. Biochemical analysis of HCT116 cells overexpressing a deacetylation mutant demonstrates an overall oxidized intracellular environment compared to overexpression of the wild‐type SIRT3 gene, as monitored by increases in intracellular superoxide and oxidized glutathione levels (Wang et al., 2007). As such, both SIRT3 and FOXO3a follow a cascade response pathway for mitochondrial signaling (Jacobs et al., 2008). Tissue fibrosis caused by aging was reported to be mediated by an enzyme called glycogen synthase kinase 3β (GSK3β) (Frame & Cohen, 2001). The deacetylation of GSK3β by SIRT3 can thus potentially block aging process associated tissue fibrosis (Sundaresan et al., 2016).

Figure 4

SIRT3 regulates aging by interacting with FOXO.

SIRT3 can also interact with the mitochondrial acetyl‐coA synthetase known as AceCS2 (Allison & Milner, 2007). SIRT3/AceCS2 is required for G1 arrest that is induced by the loss of Bcl‐2 in a specific type of noncancer epithelial cells (ARPE19 cells). This event suggests a role of SIRT3/AceCS2 in apoptosis and growth regulation under certain environmental conditions (Allison & Milner, 2007). Although the molecular basis for lifespan extension is not yet clear, up‐to‐date knowledge on SIRT3 can stimulate scientists to seek exciting therapeutic solutions for aging.

SIRT3 in energy homoeostasis

SIRT3 is an important regulator of basal ATP and controls overall energy homeostasis. Some tissues such as the heart, kidney, and liver normally express high levels of SIRT3, leading to a marked reduction in ATP in the absence of the deacetylase (Ahn et al., 2008). However, SIRT3 is capable of boosting the level of ATP in mitochondria due to the acetylation process. As mammalian mitochondrial ribosomal proteins are all nucleus‐encoded, some of them have been mapped to regions associated with disorders of mitochondrial energy metabolism (O'Brien et al., 2005). Alterations in the expression level and mutations in the genes encoding these ribosomal proteins can affect mitochondrial protein synthesis, cell growth, and apoptosis (Miller et al., 2004; Chintharlapalli et al., 2005; Yoo et al., 2005). Mitochondrial ribosomal protein L10 (MRPL10) is the major acetylated protein identified in the mitochondrial ribosome. Ribosome‐associated SIRT3 was thus found to be responsible for the NAD‐dependent deacetylation of MRPL10 (Yang et al., 2010). Mitochondrial biogenesis is also mandatory for oocyte development (John et al., 2010). In vitro maturation technique with metaphase II oocytes disclosed the developmental efficiency of SIRT3 by which mitochondrial energy homoeostasis and subsequent oocyte maturation are regulated. Hence, SiRNA‐induced SIRT3 knockdown precludes the biogenesis (Zhao et al., 2016).

SIRT3 in oxidative stress

SIRT3 also plays a significant function in the regulation of ROS levels by influencing different enzymes like SOD2, manganese dismutase, and catalase (Miao & St Clair, 2009; Merksamer et al., 2013). As increases in ROS levels can stimulate SIRT3 transcription, it may ultimately lead to deacetylation of SOD2 with the activation of oxidative stress (Chen et al., 2011); however, PGC‐1α plays a vital role in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis, and respiration to induce ROS‐detoxifying enzymes (Lin et al., 2005; Finck & Kelly, 2006). Nonetheless, the molecular mechanism underlying this phenomenon is not yet fully understood. PGC‐1α and nuclear ERRα are known to strongly stimulate gene expression of mouse SIRT3 in hepatocytes and muscle cells (Lin et al., 2005; Giralt and Villarroya, 2012).

SIRT3 in stress‐related gene expression

Human SIRT3 exists in a full‐length (FL) form that is processed to a distinct short form to localize specifically to the mitochondria; hence, only the short form of human SIRT3 is active as an NAD+‐dependent deacetylase (Schwer et al., 2002). It was reported that FL SIRT3 may also be present in the nucleus and is capable of activating histone deacetylase (HDAC) against acetylated histone H3 Lys 9 (H3K9ac) and H4K16ac in vivo (Scher et al., 2007; Nakamura et al., 2008; Sundaresan et al., 2008). Such HDAC activity is believed to be associated with transcriptional repression when artificially recruited to a transgenic reporter (Tao et al., 2010). As nuclear FL SIRT3 is subject to cellular stress conditions (e.g., oxidative stress and UV irradiation), it can lead to rapid degradation without affecting the mitochondrial SIRT3 short form. Therefore, the rapid removal of SIRT3 from chromatin could induce genes required for the stress response. Binding of FL SIRT3 to chromatin could also suppress neighboring genes and mediate the deacetylation of H4K16 by activating ubiquitination and proteasome degradation (Iwahara et al., 2012). However, there have been some contradictions about the localization of SIRT3. Many reports nonetheless claimed that SIRT3 should be exclusively located at mitochondria (Michishita et al., 2005; Lombard et al., 2007; Cooper & Spelbrink, 2008).

SIRT3 in thermogenesis

SIRT3 is also expressed in brown adipose tissue (BAT), where it is localized to the inner membranes of mitochondria (Shi et al., 2005). Caloric restriction activates SIRT3 expression in both BAT and brown adipose tissue (WAT). Unlike BAT, SIRT3 gene expression does not occur in WAT upon cold exposure. In HIB1B brown adipocytes, an imposed expression of SIRT3 augments the expression of PGC‐1α, uncoupling protein 1 (UCP1), and a series of mitochondria‐related genes in the presence of both ADP‐ribosyltransferase and the deacetylase activity of SIRT3 (Shi et al., 2005). A SIRT3 deacetylase mutant has a synergistic effect with PGC‐1α to activate UCP1 expression in the mitochondrial inner membrane to mediate the process of adaptive thermogenesis. In addition, SIRT3 stimulates CREB phosphorylation to directly activate the PGC‐1α promoter. Sustained expression of SIRT3 decreases ROS production and membrane potential, while causing increased cellular respiration. SIRT3 and other genes controlling mitochondrial function are downregulated in the BAT of several genetically obese mice (Shi et al., 2005). In the absence of SIRT3, high levels of fatty acid oxidation intermediate products and triglycerides are formed, while long‐chain acyl‐CoA dehydrogenase (LCAD) is hyperacetylated at Lys 42. LCAD can also be deacetylated under fasting conditions by SIRT3 as hyperacetylation of LCAD reduces its enzymatic activity (Allison & Milner, 2007; Hirschey et al., 2010).

SIRT3 in tumor suppression

To date, the most remarkable feature of SIRT3 is its ability to suppress tumors and/or cancer. As a key regulator of different cancers, SIRT3 detoxifies ROS to act as a tumor suppressor (Schumacker, 2010; Tanno et al., 2012; Tseng et al., 2013). Under such conditions, mitochondrial MnSOD is an important antioxidant enzyme that is primarily regulated by transcriptional activation (Miao & St Clair, 2009). Decrease in enzymatic activity is caused by acetylation at Lys 68; however, mitochondrial deacetylase SIRT3 is able to bind and deacetylate for its activation. Increase in ROS levels also stimulates SIRT3 transcription, leading to SOD deacetylation and activation. Thus, SOD‐mediated ROS reduction is synergistically increased by SIRT3 co‐expression, although it can be negated by SIRT3 depletion. As a result, a mechanism involving post‐translational regulation of SOD activity was revealed by elaborating the effect to oxidative stress on acetylation and SIRT3‐dependent deacetylation (Chen et al., 2011). However, deletion of SIRT3 removes the requirement for the loss of a tumor suppressor for transformation of primary cells with an oncogene (Kim et al., 2010). SIRT3 KO mouse embryonic fibroblast (SIRT3−/− MEF) demonstrates genomic instability as well as aberrant mitochondrial physiology. Single oncogenic expression (Myc or Ras) alters intracellular metabolism which is recovered by SOD (Kim et al., 2010). Contribution of SIRT3 in cancer genetics is discussed extensively in the following segment.

The role of SIRT3 in cancer

The role of SIRT3 in human cancer has been studied intensively to validate its effect on the disease state. SIRT3 has the ability to reprogram cellular metabolism. In tumor cells, glycolysis occurs at a high rate even in the presence of O2, which is known as the Warburg effect (Haigis et al., 2012). Warburg effect helps tumor cells by supplementing with biomass‐generating substrates (Finley et al., 2011a). SIRT3 regulates the Warburg effect by decreasing the high glycolysis rate in tumor cells. Restricting tumorous cells from gaining substrates and/or biomass is achieved by the decreased level of glycolysis (Finley et al. 2011). Some of the important activities of SIRT3 in different cancers are discussed here and are overviewed in Table 2 and Fig. 5.

Table 2

Function of SIRT3 in different cancers

Type of cancer	Type of cells used	Activity of SIRT3	References
Oral cancer	Oral squamous cell carcinoma cell line	Cell growth and proliferation are inhibited by SIRT3 downregulation	(Alhazzazi et al., 2011a,b)
Breast cancer	MCF‐7 cell line produced through continuous selective culture in the presence of Tamoxifen	SIRT3 decreases apoptosis and decreases cellular sensitivity to Tamoxifen	(Zhang et al., 2013b)
Hepatocellular carcinoma	Hepatoma cells	Overexpression of SIRT3 leads to JNK activation and resulting apoptosis	(Zhang & Zhou, 2012)
Esophageal cancer	Patients with esophageal cancer	Increase in SIRT3 decreases ROS levels, prolonging survival rate	(Zhao et al., 2013)
Lung cancer	Nonsmall cell lung carcinoma	Low SIRT3 decreases cancer cell growth	(Li et al., 2013)
Gastric cancer	MGC‐803 gastric cancer cells	Presence of SIRT3 decreases cancer	(Huang et al., 2014)
Bladder cancer	p53 bladder carcinoma cells	SIRT3 prevents cancer cells from spreading	(Li et al., 2010)

Figure 5

SIRT3 has both oncogenic and tumor suppressive activities.

Oral cancer

According to a tissue microarray analysis, SIRT3 was overexpressed in oral squamous cell carcinoma (OSCC) relative to normal human oral keratinocytes cell lines (Alhazzazi et al., 2011a). Cell growth and proliferation are inhibited by a downregulation of SIRT3, while OSCC cell sensitivity increases with radiation and cisplatin treatments in vitro. SIRT3 downregulation reduces tumor burden in vivo (Alhazzazi et al., 2011a); however, significant decreases in SIRT3 mRNA and protein level were due to the effect of (‐)‐epigallocatechin‐3‐gallate, an antioxidative catechin commonly found in green tea (Tao & Lambert, 2014).

Breast cancer

Tamoxifen (Tam) is commonly used as an adjuvant therapy during the management of breast cancer as it acts as an antagonist to the related selective estrogen receptor, that is, estrogen receptor‐positive (ER+). A study of the Tam‐resistant human breast cancer cell line (MTR‐3) revealed significant upregulation of SIRT3 at both the mRNA and protein levels (Zhang et al., 2013b). The above cell line was derived from the standard MCF‐7 cell line through a selective culture process in the presence of 1 mM Tam. SIRT3 level also rapidly increases in MCF‐7 cells after exposure to Tam. When the SIRT3 gene is overexpressed in MCF‐7 cells, a decrease in cellular sensitivity to Tam is accompanied by a blockage in Tam‐induced apoptosis. Furthermore, cells are susceptible to Tam and apoptotic cell death when SIRT3 expression is knocked down in MTR‐3 cells. These MTR‐3 cells also showed increases in the mitochondrial content of ERb, ROS levels, and apoptosis (Zhang et al., 2013b). In addition to ER+ breast cancer, low SIRT3 expression is also associated with survival in the ER−, HER2+, and basal breast cancer subtypes. These results imply that SIRT3 can be used as a molecular biomarker to diagnose patients with high‐risk breast cancer (Desouki et al., 2014). Although some other sirtuin molecules like SIRT7 have impacts on breast cancer (Ashraf et al., 2006), little is known about the contribution of SIRT3. A form of human cancer known as luminal B breast cancer is suspected to have certain linkage with the inefficiency of SIRT3. SIRT3 KO murine models are found to develop tumors with similar characteristics of human luminal B breast cancer due probably to acetylation of MnSOD. In contrast, wild‐type mice did not show such symptoms, while MnSOD was deacetylated by SIRT3 (Zou et al., 2016).

Hepatocellular carcinoma

SIRT3 protein expression in hepatocellular carcinoma (HCC) is associated with the degree of differentiation, pathological features, and complications with portal vein tumor thrombus. It is known that relative SIRT3 protein expression gradually decreases with the increases in differentiation between hepatoma cells and between the deterioration and progression of hepatocellular cells (Zhang et al., 2013a). The SIRT3 mechanism should thus be related to ROS levels in the mitochondria (Chen et al., 2011; Park et al., 2011; Schumacker, 2011); however, low SIRT3 expression was reported to act as a poor independent prognostic factor of survival in patients with postsurgical hepatocellular carcinoma (Zhang et al., 2012). The results of other research have provided evidence of increased recurrence with a decrease in SIRT3 level in HCC (Wang et al., 2014a). In hepatocellular carcinoma cells, overexpression of SIRT3 facilitated the activation of the JNK signaling pathway, resulting in apoptosis (Zhang & Zhou, 2012); however, low SIRT3 expression is associated with a markedly shorter period of clinical recurrence.

Esophageal cancer

A number of studies have analyzed the clinical significance of SIRT3 expression in esophageal squamous cell carcinoma (ESCC). High SIRT3 expression levels in ESCC tissues were previously reported to occur more frequently than in adjacent nonmalignant esophageal mucosa tissues (Yan et al., 2014). The 5‐year postoperational survival rate of patients with esophageal cancer was examined in relation with the SIRT3 expression (Zhao et al., 2013). Accordingly, high expression of SIRT3 was related to a shorter survival time in patients with esophageal cancer, which might be due to the suppression of apoptosis signals that prolonged the survival rate of esophageal cancer. Moreover, tumors with high SIRT3 level might lead to genomic or signaling deregulation, taking advantage of SIRT3 overexpression to prolong survival by decreasing ROS levels (Zhao et al., 2013). Presumably, an overexpression of SIRT3 may help cancer cells divide and grow further. In addition, a reduction in cellular ROS is suggested to activate anti‐apoptotic proteins like Bcl‐2 and Bcl‐xL (Li et al., 2004).

Lung cancer

Nonsmall cell lung carcinoma (NSCLC) is the most common lung cancer subtype encompassing approximately 85% of all such cases. Most of these patients have locally advanced or distant metastatic disease (stage III/IV) from the onset of symptoms. A 5‐year survival rate of NSCLC was reported to be less than 10% and 5% in male and female patients, respectively (Reungwetwattana et al., 2012). It was found that SIRT3 interacted with the full‐length nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) gene, causing its deacetylation to promote mitotic entry, growth, and proliferation of cultured cells in vitro and in vivo. Moreover, downregulation of SIRT3 apparently inhibited the acetylation of NMNAT2 and NAD+ synthesis enzyme activity (Yan et al., 2010). However, a lack of SIRT3 expression caused the inhibition of mitotic entry, growth, and proliferation of a NSCLC cell line, leading to apoptosis. This is actually related to energy metabolism involved in the interaction between SIRT3 and NMNAT2 (Li et al., 2013) (Fig. 6). NMNAT2 is a promising therapeutic target in lung cancer treatment.

Figure 6

Function of SIRT3 in lung cancer.

Gastric cancer

SIRT3 also plays a role in gastric cancer (GC); however, its role in the pathogenesis of GC remains unclear. It has been reported that SIRT3 expression level is inversely correlated with such factors as tumor infiltration, tumor differentiation, and tumor stage. In vitro experiments showed that the absence of SIRT3 in MGC‐803 GC cells significantly increased the expression of HIF‐1a (Yang et al., 2014); therefore, aberrantly decreased expression of SIRT3 was seen in patients with GC. SIRT3 is likely to function as a mitochondrial tumor suppressor in GC to affect the progression of patients with GC by exerting direct control on HIF‐1a. However, it is unclear whether the aberrant expression of SIRT3 in patients with GC is a causal factor, a subsequent effect of tumor progression, or if other mutations play a role in GC (Wang et al., 2015). The Notch family of proteins substantially contributes to cell behavior including cell proliferation, differentiation, and apoptosis (Vallejo et al., 2011). Notch‐1 is also overexpressed in GC, suggesting its critical role in the disease (Du et al., 2014). SIRT3 suppresses the proliferation of GC cells through the downregulation of Notch‐1, which suggests a novel therapeutic target in GC therapy (Wang et al., 2015). Previously, it was observed that GC patients with SIRT3 expression have better prognosis than those without it. SIRT3 expression is an independent prognostic marker of survival to act as a tumor suppressor in GC (Huang et al., 2014).

Bladder cancer

Extracellular/intracellular stress may lead to cell cycle arrest, apoptosis, and cellular senescence in bladder cancer (Li et al., 2012). Accordingly, tumor protein p53 functions to regulate the transcriptional program in the related events of above cellular stress. The mitochondrial p53 gene has an antiproliferative function, although it is normally expressed in the nucleus (Talos et al., 2005). In the absence of p53, an EJ bladder carcinoma cell line maintained growth arrest until p53 was introduced (Sugrue et al., 1997). Endogenous p53 can be independently partitioned with mitochondrial and nuclear proteins, as it is capable of promoting senescence. SIRT3 thus rescued p53‐EJ cells from induced p53‐mediated growth arrest and partially nullified the ability of p53 to enact growth arrest and senescence; however, after the introduction of the chaperone protein BAG‐2, SIRT3 targeting of p53‐mediated senescence was averted (Li et al., 2010).

The role of SIRT3 in other diseases

Although SIRT3 has a potential role in cancer, it can also exert important control on other diseases. The most notable aspects of these diseases are described below.

Alzheimer's disease

ROS marker analysis revealed the prevalence of oxidative stress in Alzheimer's disease (AD). This analysis was carried out by determining oxidized biomolecules generated by ROS (Wang et al., 2014b). Oxidative stress is engaged in a spiral interaction with mitochondrial dysfunction. Thus, it poses a major threat by producing ROS that are critical for the pathogenesis of AD as well as a number of other neurodegenerative diseases (Lin & Beal, 2006). Recent studies revealed that SIRT3 expression has been increasing in both human and mouse AD pathology. Apolipoprotein E4 (APOE4) is a major genetic factor in late‐onset AD, and SIRT3 is known to be downregulated in the frontal cortices of human brain APOE4 carriers compared to noncarriers. Analysis of SIRT3 level may aid in the diagnosis of AD (Yin et al., 2015). In addition, SIRT3 level may increase in response to increased ROS synthesis. In an experiment with primary neuronal cultures, the occurrence of such SIRT3 upregulation indicated a possibility for an increased oxidative stress‐oriented mitochondrial response in AD. An increase in LF SIRT3 could well extend the neuronal lifespan under mitochondrial oxidative stress (Weir et al., 2012).

Huntington's disease

Huntington's disease (HD) as a progressive and autosomal dominant neurodegenerative disorder develops with psychiatric manifestations, cognitive decline, and movement abnormalities (Harper, 1996). HD is caused by abnormal polyglutamine expression in the Huntingtin (Htt) protein (Naia & Rego, 2015). To date, there is no cure for HD. Nonetheless, as energy‐demanding neurons are particularly susceptible to energy deficits and oxidative stress, mitochondrial dysfunction might be a mediating factor for the mutant Htt‐induced neurotoxicity (Fu et al., 2012). It was previously reported that cells expressing mutant Htt displayed reduced SIRT3 levels, and mutant Htt‐induced depletion of SIRT3 protected cells from mutant Htt. The natural product viniferin and other semisynthetic stilbenic compounds can also decrease ROS levels; as such, they will decrease deacetylase activity along with a reduction in cellular NAD+ levels and mitochondrial biogenesis in cells. Viniferin also activates AMP‐activated kinase while enhancing mitochondrial biogenesis. On the other hand, a knockdown significantly inhibited viniferin‐mediated AMP‐activated kinase activation with diminished neuroprotective effects (Fu et al., 2012). Thus, SIRT3 is suggested to mediate the neuroprotection of viniferin (Fu et al., 2012).

Amyotrophic lateral sclerosis

The ALS disease shows an increase in fragmented mitochondria along with defects to bi‐directional axonal transport with increased cell death. In a model of amyotrophic lateral sclerosis (ALS), SIRT3 is able to prevent mitochondrial fragmentation of spinal cord motor neurons transfected with SOD1G93A. Interestingly, co‐expression with either SIRT3 or PGC‐1α is able to rescue SOD1G93A‐induced mitochondrial fragmentation to improve cell survival (Song et al., 2013). SIRT3 regulates ketone body production by deacetylation of mitochondrial 3‐hydroxy‐3‐methylglutaryl CoA synthase 2 (HMGCS2), indicating its potential for neuroprotection in the SOD1G93A model (Pasinetti et al., 2013).

Age‐related hearing loss

Age‐related hearing loss (AHL) is frequently explained in association with oxidative stress. In mice, oxidative stress causes damage to hair cells and spiral ganglia neurons of the cochlea, which results in hearing loss in about 12 months. Calorie restriction induces SIRT3, which prevents or delays age‐related hearing loss by protecting cochlear neurons from oxidative damage (Someya et al., 2010; Bell & Guarente, 2011). Mitochondria can maintain oxidative stress by the glutathione antioxidant system that is mediated by increased levels of NADPH, which is achieved by direct deacetylation of mitochondrial isocitrate dehydrogenase 2 (IDH2) by SIRT3 (Someya et al., 2010; Jing et al., 2011).

Conclusion

SIRT3 is a NAD+‐dependent protein deacetylase. It is a noble protein with numerous roles that are essential in human physiology. Starting with energy homeostasis and metabolism, SIRT3 impacts nuclear and muscular function, reduces the effects of aging, and mediates several genetic diseases. Moreover, it has the ability to repair DNA, regulate thermogenesis, and fight against oxidative stress. SIRT3 has been noted extensively for its role in different types of cancer such as oral cancer, breast cancer, esophageal cancer, lung cancer, gastric cancer, and hepatocellular carcinoma and also in other diseases such as AD, HD, ALS, and AHL. Perceptual research in the field of SIRT3 is continually increasing our knowledge. Both in vitro and in vivo studies can be used to acquire information regarding the potential of different SIRT3 activators and the pharmacological applications of this protein. In summary, SIRT3 is a clinically novel target for various complications of human physiology.

Conflict of Interest

The authors have no conflict of interests to declare.