Effects of Calorie Restriction and Sirtuin Activation for Increasing Longevity 

CALORIE RESTRICTION AND METABOLISM

Metabolic changes during starvation

Starvation initiates drastic changes in metabolism and metabolic regulation. Some of the major metabolic processes that are altered during periods of starvation include gluconeogenesis, ketogenesis, glycogen mobilization, and fat mobilization. Changes in hormone levels, such as insulin, glucagon, adipokines, and glucocorticoids, are also altered as a result. These observed responses occur because of adaptations that have been put in place to preserve glucose for the brain (Rodgers, 2005). Since glucose is the preferred energy source for the brain and many other organ systems, it is essential to have safeguards put in place to maintain adequate glucose levels.

When starvation occurs, low blood glucose levels lead to a decreased secretion of insulin and an increased level of glucagon, which induces gluconeogenesis and glycogenolysis. These effects lead to the mobilization of triglycerides from adipose tissue to produce metabolic intermediates that can feed into the Krebs Cycle. When starvation persists, glycogen storages are depleted and ketone bodies are then produced to serve as an energy source.

These safeguards that are initiated during times of starvation allude to the fact that CR may be responsible for promoting longevity by regulating metabolism and providing alternative energy sources when nutrients are low.

Early Hypotheses for Calorie Restriction Increasing Longevity

From an evolutionary standpoint, these mechanisms were crucial for the survival of organisms during periods of limited food sources. It is believed that these sources of regulation evolved to respond to the environment and promote cellular defense mechanisms in instances of adversity by boosting energy production (Dai, 2018).

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About CR, there is a possibility that the effects of the observed improvements in longevity occur due to mimicry of the cellular defense mechanisms resulting from starvation. As mentioned previously, one of the early hypotheses suggested that CR increased longevity by simply reducing the metabolic rate. The reasoning behind this was due in part to the decreased amounts of oxygen radicals.

Reactive oxygen species (ROS) are generated during the electron transport chain of cellular respiration. ROSs are highly reactive and induce severe damage to cellular structures, which in turn contributes to the aging process over time. It was believed then that a reduced metabolic rate from CR simply reduced the amount of ROSs produced and thus delayed the aging process (Harmon, 1956). This hypothesis was ultimately proved incorrect when it was discovered that metabolic rate did not decline in CR mice and that respiration increased in CR yeast (Lin, 2000). It was shown that a decrease in glucose affected the flux in carbon metabolism and switched it towards the Krebs cycle, thus increasing cellular respiration (Figure 1). The rejection of this early hypothesis prompted researchers to direct their focus towards Sirtuins, a much more promising explanation for the observed increases in lifespan resulting from CR.

EFFECTS OF CALORIE RESTRICTION AND SIRTUINS ON INCREASED LIFESPAN

Silent Information Regulator 2 Gene

The Silent Information Regulator 2 (Sir2) gene in Saccharomyces cerevisiae was one of the first genes discovered to extend lifespan (Imai, 2000). This gene was shown to have gene silencing activity as well as NAD-dependent histone deacetylase activity of target lysine residues. Histone deacetylases are enzymes with chromatin-remodeling capabilities that can silence gene transcription through chromatin compaction (Figure 2). Sir2 requires NAD+, which is a cofactor that is also involved in redox reactions and can act as a nutrient sensor (Denu, 2005). The discovery of the histone deacetylase activity in Sir2 suggested that Sir2 might also act as a nutrient sensor that can alter gene transcription in times of metabolic stress (Imai, 2000).

In an early study examining Sir2 effects on longevity, Sir2 dictated replicative lifespan in the yeast, which was observed by the number of daughter cells a mother cell could produce (figure 3). CR extended the lifespan in the yeast and showed upregulation in the silencing activity by Sir2. Thus, increased lifespan from CR was shown to require Sir2 regulation and was accompanied by an increase in respiration and Sir2 activity (Kaeberlein, 1999). Since NAD+ is ubiquitous for metabolic regulation, this initial finding suggested that Sir2 might be an important aspect in connecting cellular energetics to lifespan (Kaeberlein, 1999). From this point on, studies were aimed to discover if Sir2 and its homologs can act as a nutrient sensor to relay changes in NAD+/NADH ratios and ultimately alter gene transcription. Sir2 has since been known to be an evolutionarily conserved gene. Sirtuins in species ranging from fruit flies to worms have subsequently been discovered, due to their homology to Sir2.

Mammalian Sirtuin Orthologs

The findings of Sir2 and sirtuin variants in other species generated interest in searching for a mammalian sirtuin. It was found that there are seven sirtuins (SIRT1-7) in mammals, many of which displayed similar functions as Sir2 (Bordone, 2005). Specifically, SIRT1 was shown to possess NAD-dependent deacetylase activity and was similar to the activity of Sir2 in yeast (Oberdoerffer, 2008). The SIRT family of genes is involved in regulation at many levels, including transcription, translation, protein stability, and oxidation. These sirtuins are regulated through inhibition, protein-protein interactions, microRNAs, localization within organelles, and by substrate availability (Oberdoerffer, 2008). The diversity of regulatory pathways that sirtuins are involved in presents issues for pinpointing the exact roles and relation to improved longevity. Nonetheless, these genes display promising characteristics for understanding the link between CR and longevity.

SIRT 1, SIRT6, and SIRT 7 are involved in transcription regulation by acting on transcription factors, which can act as controls for energy metabolism, cell survival, DNA repair, tissue regeneration, and neuronal signaling (Haigis, 2010). Of these three sirtuins, SIRT1 resembled Sir2 the closest, due to its deacetylase activity. SIRT1 has also been found to have expression across all mammalian somatic and germ cells, making it an attractive candidate for studying the association between CR and longevity.

Interactions of Sirtuins with Conserved Longevity Pathways

It is known that sirtuins play a role in a large number of conserved pathways that determine longevity. Examples of such pathways include AMP-activated protein kinase (AMPK) and insulin and insulin-like growth factor 1 (IGF1) signaling, which subsequently targets IGF1, mechanistic Target of Rapamycin (mTOR), and forkhead box O (FOXO) (Motta, 2004). The interactions of sirtuins and these major pathways show the versatility in their mechanisms of action and illustrate that their effects could play a critical role in regulating the aging process (Figure 4).

For the first pathway mentioned, AMPK is a key regulator of energy metabolism and responds to variation in the ratio of ATP to AMP. There are various interactions between SIRT1 and AMPK, which regulate NAD+/NADH ratios and ATP/AMP ratios respectively (Wang, 2011). The cross-regulation of these energetic pathways was shown to affect the development and aging of endothelial tissues. When SIRT1 was unregulated in the endothelium of mice, there was increased protection from premature vascular deterioration (Wang, 2011).

Additionally, sirtuins are also involved in insulin signaling pathways. Reduced levels of insulin and IGF1 signaling were shown to be a key regulator of longevity in C. elegans and Drosophila (Longo, 2009). These studies indicate that this signal reduction protects against oxidative damage and other forms of stress. SIRT1 linkage to this pathway serves as a stress resistance transcription factor. Increased levels of SIRT1, resulting from CR, can be attenuated by IGF1. Treatment of cells with insulin or IGF1 lowered SIRT1 levels, which suggests an inverse relationship between SIRT1 and the insulin and IGF1 pathway (Longo, 2009). SIRT1 also increases the release of insulin and further affects cellular insulin sensitivity. Knockout mice without SIRT1 showed an increase in the expression of IGF-binding proteins (IGFBPs), a known inhibitor of IGF1 (Bonkowski, 2016). These results relate to how insulin and glucose availability are interconnected to longevity pathways through regulation by SIRT1.

The target of Rapamycin (TOR) is also involved in the observed effects of CR through regulation by sirtuins (Figure 4). TOR is a serine and threonine protein kinase that regulates cell growth through the regulation of protein synthesis and transcription. Upstream signaling from insulin and IGF1 allows TOR to act as a nutrient, energy, and redox sensor. Following the connection between IGF1 and now TOR, studies have found that TOR inhibition extends lifespan in yeast by increasing Sir2 activity (Ghosh, 2010). Stress conditions downregulate TOR signaling, which reduces protein synthesis and cell proliferation. This effect is counteracted in the absence of SIRT1 in mice. Recent studies have used inhibitors of TOR as drug therapy for treating insulin-related diseases. Rapamycin, an inhibitor of TOR, was shown to increase the lifespan of mice (Mercken, 2012). It has been thought that Rapamycin increases longevity by mimicking diets that have low levels of essential amino acids, like methionine or tryptophan. Additionally, Resveratrol acts as a SIRT1 activator and was shown to inhibit TOR activity and prevent cellular senescence

Another noteworthy target of sirtuin-mediated deacetylation is the FOXO transcription factor. FOXO and its orthologs are highly conserved transcription factors across many species and play a critical role in longevity, metabolism, and stress responses. In C. elegans, it was found that DAF-16 (a FOXO ortholog) selectively regulates the transcription of many genes and ultimately improved immunity and extended lifespan (Rizkai, 2011). The observed FOXO responses are initiated through a kinase cascade and phosphorylation resulting from the simulation of the insulin and IGF1 pathway. The connection between FOXO, IGF, and the previously discussed role of CRthat  in this network, provides several notable studiesthat that have further evidence to support the notion that CR may lead to increased longevity (Figure 4).

Calorie Restriction and Sirtuins

Many experiments have been conducted across various species to test for the relationship between CR and sirtuins (Table 1). It is known that CR leads to changes in NAD+/NADH levels and sirtuin activity (Haigis, 2006). Furthermore, it is believed that sirtuins are the main reason behind improved health from CR and exercise. Although these results appear promising, it is still unclear as to what the exact connections are that link these two processes to increased longevity (Figure 5). The main goal of recent studies has been to determine this relationship to develop drug therapies for humans. There are several notable studies that have been previously conducted that lay the foundation for linking CR and sirtuin regulation, but clear conclusions have still been unattainable.

For example, a strain of transgenic mice with overexpression of SIRT 1 displayed many of the common outcomes associated with CR, including increased metabolic activity, reduced blood lipid levels, and improved glucose metabolism, but failed to prove a definite increase in lifespan (Bordone, 2005). A different strain of transgenic mice with moderate overexpression of SIRT1 showed better protection from inflammation, liver cancer, diabetes, and hepatic steatosis, but also did not have increased longevity (Sohal, 1996). To advance the understanding of these effects, it is essential to conduct more research to further understand how sirtuins are activated. Achieving this missing element can potentially fill in the gaps and create new therapeutic methods for treating age-related diseases.

Sirtuin Activating Compounds

The search for molecules with the ability to activate sirtuins began over a decade ago. Resveratrol, the first known STAC for SIRT1, was discovered in 2003 (Howitz, 2003). This discovery played a critical role in determining that sirtuins can be regulated through allosteric activation. While most enzyme-targeting drugs function through inhibition, molecules with the ability to bind to allosteric sites can act through activation. Development of such activating molecules can be challenging, but they are advantageous in the fact that they have a high level of specificity and induce fewer side effects than inhibitory drugs (Dai, 2018). STACs increase the binding affinity of SIRT1 to its substrate NAD+, which in turn increases enzymatic activity tenfold (Howitz, 2003). Extensive structural studies found that STACs bind to a Sirtuin Binding Domain (SBD), which sequesters the STAC-bound SBD closer to the SIRT1 active site to improve substrate binding (Dai, 2018). The crystal structure of STACs was further analyzed and it was determined that Resveratrol functions by binding to a rigid helix-turn-helix region on the N-terminus of SIRT1. The N-terminus was found to be a key mediator in the allosteric activation of SIRT1 by STACs (Howitz, 2003).

Using this information, high-throughput screening and fluorescence-based screens have been used to elucidate that SIRT1 prefers STACs with specific hydrophobic amino acids in locations adjacent to an acetylated lysine residue on SIRT1. Since these pivotal discoveries, more than 100 synthetic STACs have been identified, indicating that SIRT1 can be activated by both natural and synthetic STACs (Hubbard, 2013). Combining the known mechanisms of STAC activation of sirtuins and the connected longevity pathways mediated by sirtuin activity, there is now enough information to fully investigate how CR ultimately leads to increased longevity. The next step for many of the current research is to use this knowledge and apply it to primate and human studies to find new drug treatments for insulin-related illnesses and age-related diseases, which will be discussed later in this review.