Many researchers, over years of studies, have come to believe that the answer to what makes resveratrol so potent lies in understanding its interaction with the sirtuin gene family. Of the seven sirtuin genes known in humans, the SIRT1 gene is perhaps the best known and studied for producing enzymes that appear to increase DNA stability and protect against cellular degenerative processes throughout the body, but scientists continue to gain a more thorough understanding about the role of other sirtuins in human health every year.
Typically sirtuin genes are activated when the cell or body is under some sort of biological stress, like that of calorie-restriction. But a more accurate view of sirtuin genes is that they are intricately connected to metabolic cellular activity—regulating the production of enzymes, hormones, and other proteins in response to physiological conditions.
What is SIRT-1?
Sirtuin 1 (SIRT1) is an evolutionarily conserved NAD+-dependent deacetylase that is at the pinnacle of metabolic control, all the way from yeast to humans. SIRT1 senses changes in intracellular NAD+ levels, which reflect energy level, and uses this information to adapt the cellular energy output such that it matches cellular energy requirements. The changes induced by SIRT1 activation are generally (but not exclusively) transcriptional in nature and are related to an increase in mitochondrial metabolism and antioxidant protection. These attractive features have validated SIRT1 as a therapeutic target in the management of metabolic disease and prompted an intensive search to identify pharmacological SIRT1 activators. In this review, we first give an overview of the SIRT1 biology with a particular focus on its role in metabolic control. We then analyze the pros and cons of the current strategies used to activate SIRT1 and explore the emerging evidence indicating that modulation of NAD+ levels could provide an effective way to achieve such goals.
During the last decade the mammalian sirtuin (SIRT1) family (formed by paralogs SIRT1–SIRT7) has emerged as a constellation of enzymes with key roles in whole-body metabolic homeostasis and an interesting therapeutic potential applicable to multiple pathophysiological states.
The history of sirtuins began almost 3 decades ago, with the identification of Sir2 (silent information regulator 2), a protein-forming part of a complex that enabled gene silencing at selected regions of the yeast genome (Shore et al., 1984; Ivy et al., 1986). A major turning point in the history of Sir2 came from the discovery that Sir2 was involved in the yeast replicative aging process (Kaeberlein et al., 1999). The accumulation of extrachromosomal rDNA circles (ERCs) as the organism ages is believed to be a major determinant of yeast replicative lifespan (Sinclair and Guarente, 1997). Although the mechanism by which the accumulation of ERCs influences lifespan is not fully understood, different genetic manipulations promote reasonable, despite correlational, evidence that the accumulation of ERCs is negatively correlated with yeast replicative aging (Sinclair and Guarente, 1997;Defossez et al., 1999; Kaeberlein et al., 1999). It was originally thought that the impact of Sir2 on replicative lifespan of yeast was consequent to its silencing activity on ERCs. However, the effects of Sir2 on aging extend further than ERC silencing, because genetic manipulations of Sir2 orthologs can also affect lifespan of higher eukaryotes, such as the nematode Caenorhabditis elegans (Tissenbaum and Guarente, 2001; Viswanathan et al., 2005; Berdichevsky et al., 2006; Rizki et al., 2011) and insects such as Drosophila melanogaster (Rogina and Helfand, 2004; Bauer et al., 2009), where ERCs are not thought to cause aging. However, there are some caveats on the consistency, amplitude, and mammalian translation of the lifespan-extension effects of Sir2 orthologs (Kaeberlein and Powers, 2007;Burnett et al., 2011; Lombard et al., 2011; Viswanathan and Guarente, 2011), which suggest that the effects of Sir2 on organismal lifespan might be indirect and/or largely depend on a specific repertoire of third-party modulators.
If not acting primarily as lifespan determinants, what then is the exact function of this family of proteins? A first hint of the real function of Sir2, or its orthologs, was grasped when the activity of Sir2 as a silencing enzyme was more precisely defined as a NAD+-dependent deacetylase (Imai et al., 2000). In the reaction catalyzed by sirtuins, an acetylated substrate gets deacetylated, using NAD+ as a cosubstrate, and yielding the deacetylated substrate, nicotinamide, and 2′-O-acetyl-ADP-ribose (Fig. 1). The NAD+ dependence and the relatively high Km of the Sir2 enzyme for NAD+ immediately suggested a potential link between Sir2 activity and the metabolic state of the cell (Guarente, 2000).
In mammals, there are seven Sir2 orthologs (SIRT1–SIRT7) that constitute the sirtuin family of enzymes. All of them are ubiquitously expressed and share a conserved catalytic core comprising 275 amino acids (for review, see Dali-Youcef et al., 2007; Michan and Sinclair, 2007). The different members of the mammalian sirtuin family, however, show distinct features that probably endow them with specialized functions. For example, mammalian sirtuins differ in their subcellular localization. SIRT1, the best characterized family member, resides mainly in the nucleus (Michishita et al., 2005) but can shuttle from the nucleus to the cytosol (Tanno et al., 2007), where several of its targets are found. SIRT2 is localized mainly in the cytoplasm, although it can also regulate gene expression by deacetylation of transcription factors that shuttle from the cytoplasm to the nucleus (Jing et al., 2007), and it contributes to chromatin compaction upon disassembly of the cell nucleus during mitosis (Vaquero et al., 2006). SIRT3, SIRT4, and SIRT5 are generally considered mitochondrial proteins (Onyango et al., 2002;Schwer et al., 2002; Michishita et al., 2005), whereas SIRT6 and SIRT7 are nuclear proteins. However, although SIRT6 is located predominantly in the heterochromatin, SIRT7 is thought to be mainly enriched in the nucleoli (Michishita et al., 2005).
In addition to their differential cellular locations, the sirtuin family members can also be distinguished by their different enzymatic activities. SIRT1 and SIRT5 act as deacetylases (Imai et al., 2000; Vaziri et al., 2001), whereas SIRT4 seems to be a mono-ADP-ribosyl transferase (Haigis et al., 2006). SIRT2, SIRT3, and SIRT6 can display both activities (North et al., 2003; Liszt et al., 2005; Shi et al., 2005;Michishita et al., 2008). The activity of SIRT7 has not been clearly established, even though it has been hypothesized to act as a deacetylase (Vakhrusheva et al., 2008). It is noteworthy that SIRT5 was recently described to demalonylate and desuccinylate proteins (Du et al., 2011; Peng et al., 2011). It is tempting to speculate that the spectrum of action of sirtuin is not limited to deacetylation but would cover a much wider range of acylation-based post-translational modifications. The identification of sirtuin substrates during the last few decades has clearly pointed out a prominent role of sirtuins as metabolic regulators. For the purpose of this review, we mostly focus on the actions of SIRT1. For extensive discussion of the actions of other sirtuin members, we refer the reader to reviews elsewhere (Dali-Youcef et al., 2007; Michan and Sinclair, 2007; Yamamoto et al., 2007; Schwer and Verdin, 2008; Finkel et al., 2009; Guarente, 2011).
Sirtuin 1: What and Where Is It?
Among all sirtuins, SIRT1 is the best characterized. Human SIRT1 contains the conserved catalytic core of sirtuins and both N- and C-terminal extensions that all span ∼240 amino acids (Fig. 2). These extensions serve as platforms for interaction with regulatory proteins and substrates. In total, the human SIRT1 spans 747 amino acids. SIRT1 contains two nuclear localization signals as well as two nuclear exportation signals (Tanno et al., 2007). The balanced functionality of these signals determines the presence of SIRT1 in either the nuclear or the cytoplasmic compartment and explains why SIRT1 location may differ depending on the cell type or tissue evaluated. For instance, although SIRT1 is found mainly in the nuclear compartment in COS-7 cells (McBurney et al., 2003; Sakamoto et al., 2004), it is abundantly found in the cytosol of rodent β cells, myotubes, and cardiomyocytes (Moynihan et al., 2005; Tanno et al., 2007). Although the implications and regulation of SIRT1 shuttling are still largely unknown, some experiments indicate that SIRT1 shuttles from the nuclei to the cytosol upon inhibition of insulin signaling (Tanno et al., 2007). The latter observations suggested a link between SIRT1 activity and the sensing of the metabolic status of the cell, as discussed in the next chapter.
Researchers agree that it’s the resveratrol that can be credited–but while resveratrol is often found in food, it is simply not possible to get the amount of resveratrol you need to help stimulate the SIRT1 gene consuming a tremendous amount of food and red wine per day.
SIR-2 – The Longevity Enzyme
Researchers have known for decades that reducing daily intake of calories by up to 40 percent dramatically slows down the aging process and extends lifespan of lab animals. Caloric restriction also has been shown to protect mammals from cancer and other age-related diseases. Recently scientists identified a class of regulatory “longevity genes” that are shared by almost all living organisms. These genes function as a feedback system to enhance survival during times of stress, such as during drought or famine. The process begins when external signals indicate a deterioration of environmental conditions. Once triggered by environmental cues, the longevity genes “switch on” and induce defensive changes at the cellular level, such as slowing metabolism and enhancing cellular respiration to help the body adapt to a more beneficial survival program.
In their study, the Harvard researchers focused on a family of enzymes, called sirtuins, produced by almost all life forms – from single celled organisms, to plants and mammals – during times of stress, such as famine (or caloric restriction). Sirtuins (silent information regulator proteins) are known to act as guardian enzymes that protect cells and enhance cellular survival. The human sirtuin, SIRT-1, for example, has been shown to suppress the p53 enzyme system normally involved in suppressing tumor growth and instigating cell death (apoptosis). By suppressing p53 activity SIRT-1 prevents the cycle of premature aging and apoptosis normally induced when cellular DNA is damaged or stressed, thus giving cells enough time to repair any damage and prevent unnecessary cell death. A second sirtuin found in yeast, SIR2, has also been shown to become activated when placed under stress. SIR2 has been shown to increase DNA stability and speed cellular repairs, while increasing total cell lifespan.
“We think sirtuins buy cells time to repair damage,” said molecular biologist David Sinclair, assistant professor of pathology at Harvard Medical School and co-author of the new study. “There is a growing realization from the aging field that blocking cell death – as long as it doesn’t lead to cancer – extends lifespan.”
An Alternative to Caloric Restriction
Intrigued by the positive health benefits of caloric restriction, the Harvard research team began to search for other methods of modulating sirtuin activity without resorting to starvation. After an initial screening process, the researchers discovered that several plant metabolites acted as sirtuin-activating compounds (STACs). Plants produce a variety of polyphenols, such as resveratrol, flavones, stilbenes, isoflavones, catechins and tannins in response to environmental stresses, such as drought, nutrient depleted soils, ultraviolet radiation and pathogens. As they refined their screening process the researchers discovered that the most potent activator of sirtuins was resveratrol.
To test the ability of resveratrol to activate sirtuins in living creatures the Harvard researchers selected yeast, a
single-celled organism that is closely related to animals, including humans. The research team hypothesized that if resveratrol was effective in modifying the newly identified target genes to trigger sirtuin production it would closely reflect the protein’s role in animals to formally link the protein to lifespan extension, at least for yeast. Their study found that even small doses of resveratrol helped yeast cells live as much as 60 to 80 percent longer, as measured by the number of generations (Fig. 1). Yeast treated with resveratrol lived for an average of 38 generations, as compared to only 19 generations for untreated yeast.
Additional experiments with human cells found that resveratrol activated a similar pathway requiring SIRT1 that enabled 30 percent of the treated human cells to survive gamma radiation compared to 10 percent of untreated cells. In the paper, the researchers also report that preliminary experiments with flies and worms are encouraging, and mouse studies are in the works.
The fact that human sirtuin SIRT1 turns off the tumor suppressor gene p53 has raised some concern that activating the sirtuin pathway to switch on the cellular longevity program might actually promote cancer. In addressing this issue, Sinclair noted that calorie-restricted animals in experiments by others have lower, not higher rates of cancer. Additional studies also found that resveratrol is able to block all three mechanisms of cancer formation by helping the body inhibit tumor initiation, promotion and progression. Resveratrol has also been shown in numerous clinical trials to benefit heart disease by reducing platelet aggregation and increasing HDL-cholesterol. Resveratrol, in combination with other bioflavonoids and vitamins C and E, may have a synergistic effect by reducing pathological platelet aggregation, stimulating healthy blood flow via the dilation of arteries, and minimizing free radical damage to blood vessel linings.
Research indicates that plant polyphenols, which increase in plants in response to stressful conditions, (similar to conditions found in humans while dieting), help to cue organisms to prepare for impending harsh conditions by switching to a more beneficial survival program. The plant polyphenol, resveratrol, has been shown to act in just such a manner to activate sirtuins to mimic the benefits of caloric restriction to slow aging and extend lifespan – in the case of yeast, up to 80 percent beyond untreated samples. Furthermore, resveratrol has been shown to activate sirtuins, which are also active in human cells, suggesting a potential for lengthening life and preventing or treating aging-related diseases in humans.
Trans- and Cis-Resveratrol
Resveratrol, found in the skins of young unripe red grapes, occurs naturally in two related forms, or isomers, referred to as trans-resveratrol and cis-transrevatrol. Of the two, the trans form (3,5,4′-trihydroxy-trans-stilbene), is the one that has been shown in numerous studies to be the most bioactive and clinically beneficial form of resveratrol. Recommend doses range from 5 mg of trans-resveratrol daily for preventive purposes up to 20 mg, twice daily, for therapeutic purposes.