Review Article 2013

Settling the Debate: Hypothalamic SIRT1 as the Key to Longevity

Abstract

SIRT1 has received a lot of attention given that it is the homolog in mammals of the silent mating type-2 (Sir2) in yeast, a protein that is in charge of regulating life span and aging. This notion has catapulted in extensive research regarding the characterization of the longevity regulating role and function of SIRT1 in mammals. SIRT1 targets specific non-histone substrates like PGC-1a and FOX proteins to regulate systemic and cellular adaptation in response to various types of stresses. Their activation enhances mitochondrial biogenesis and activity by means of gluconeogenesis, which has been related to longevity because instead of going through apoptosis, the cells survive. Due to many conflicting results in studies, the idea that SIRT1 could be the key to longevity has become very controversial. Recent studies that have analyzed the role of SIRT1 in response to oxidative stress such as caloric restriction have published some interesting results. According to one these studies, when SIRT1 is overexpressed in certain areas of the hypothalamus, like the ARC and PV nuclei, instead of enhancing longevity it causes weight gain and obesity. On the other hand, another studied proposed that overexpressed SIRT1 in the DMH and LH nuclei of the hypothalamus suppresses the activity of the ARC and PV nuclei and, through a novel SIRT1/Nkx2-1/Ox2r pathway, SIRT1 enhances skeletal muscle function, better sleep and various other functions characteristic of a youthful physiology. In other words, this study successfully linked

SIRT1 to longevity, providing with novel findings to settle the intense debate of SIRT1 as the key to longevity. Aging, exercise, and low nutritional availability will be discussed as factors that up regulate the enzymatic activity of SIRT1 in various tissues and its effects. This review will summarize some of the most recent research studies that strive to link SIRT1 to longevity, highlight controversies, and analyze why some approaches and techniques have failed to prove this connection, while others have been successful.

Introduction

Sirtuins are a family of proteins that have been identified in various animal species. They have been identified in animals as simple as yeast and as complex as humans (Kelly 2010). They are one of the most conserved family proteins. They have been conserved through a long line of animal species indicating they must play a very important role in organisms; one that cannot be accomplished by any other molecule. By nature, sirtuins are NAD+-dependent deacetylase proteins (Kelly 2010). In other words, Sirtuins depend on the presence of NAD+ in order to carry out in their target substrates the deacetylation or removal of an acetyl group from specific amino acids.

The first component of the Sirtuin genes that code for Sirtuin proteins was identified in yeast and named silent mating type information-2 (Sir2). In yeast, Sir2 regulates various metabolic pathways including life span and aging regulation (Kelly 2010; LaGuire and Reaves 2013). Subsequent research identified a homologous gene in

Settling the debate: Hypothalamic SIRT1 as key to longevity Page 1

Paola Caballero, Chemistry Department, RISE Program UPR Cayey

Review Article 2013

mammals. Silent mating type information-2 homolog (SIRT1) in mammals is equivalent to Sir2 in yeast. Inductive reasoning led to the idea that SIRT1 could also be involved in the regulation of life span and aging. This notion has catapulted in extensive research regarding the characterization of the longevity regulating role and function of SIRT1 in mammals. Due to many conflicting results in studies, the idea that SIRT1 could be the key to longevity has become very controversial. This review will summarize some of the most recent research studies that strive to link SIRT1 to longevity, highlight controversies, and analyze why some approaches and techniques have failed to prove this connection, while others have succeeded.

Sirtuin 1

In essence, SIRT1 is a deacetylase protein mainly found in the nucleus of cells, but also possibly present in the cytoplasm. It is greatly expressed in the hypothalamus, heart, kidney, liver, and skeletal muscle among others (Kelly 2010). Sirtuins are characterized for deacetylating histones, which in turn silences the expression of some genes. This was the first characterization of the regulation path of sirtuins. Nevertheless, subsequent research has established that sirtuins also target non-histone substrates (Kelly 2010; Houtkooper et al. 2012). SIRT1 has the capability of silencing histones, but its primary role is the regulation of non-histone substrates like transcription factors. By regulating transcription factors, SIRT1 has control over what genes are expressed or not. In 2009, SIRT1 was found to interact with MyoD to control the transcription of PGC-1a in skeletal muscle (Amat et al. 2009). Insights

on how SIRT1 protein directly regulates the metabolism of skeletal muscle were evidenced through this study. According to its results, SIRT1 up-regulates the promoter activity of the Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a), a transcriptional co-activator that is responsible for the expression of important genes involved in energy metabolism and mitochondrial biogenesis in different tissues, such as skeletal muscle. The myogenic determining factor (MyoD) was also shown to significantly enhance and mediate the previously mentioned positive regulation of PGC-1a by Sirt1. Evidence indicates the SIRT1/ MyoD/ PGC-1a pathway is possibly due to the deacetylase nature of the SIRT1 protein (Amat et al. 2009). Identifying this pathway is an important contribution to science for two reasons. First, the pathway aids in an understanding of SIRT1 protein’s regulatory role in gene expression, and secondly, it may be a potential regulator of the aging process as well. Both of these topics tend to generate intense debate.

Another important set of transcription factors regulated by SIRT1 is the Forkhead Box (FOX) family proteins. FOX01 protein regulates the expression of major hepatic gluconeogenic genes through the thyroid hormone (TH) and its nuclear receptor (Singh et al. 2013). When FOX01 is deacetylated by SIRT1, its transcriptional function is activated. In its deacetylated form, FOX01 activates the nuclear receptor of TH inducing TH signaling to activate the gluconeogenic genes such as phosphoenol pyruvate carboxykinase (PCK1) and glucose-6-phosphate (G6PC). SIRT1 was also found to work with transcription factor FOX03 when cells were exposed to

Settling the debate: Hypothalamic SIRT1 as key to longevity Page 2

Review Article 2013

oxidative stress (LaGuire and Reaves 2013). In this situation, the deacetylation of FOX03 induces cell cycle arrest. The acetylated form of FOX03 actually induces cell death (apoptosis). In other words, SIRT1 protects cells against apoptosis by deacetylating FOX proteins that activate genes in order to regain homeostasis.

In summary, SIRT1 targets specific non-histone substrates like PGC-1a and FOX proteins to regulate systemic and cellular adaptation in response to various types of stresses. The activation of the previously mentioned substrates through SIRT1-mediated deacetylation enhances mitochondrial biogenesis and activity by means of gluconeogenesis. Gluconeogenesis is the process by which glucose forms other compounds like amino acids, glycerol and fatty acids in order to maintain the citric acid cycle functioning (Reece et al. 2011; Houtkooper et al. 2012).

Now, aging, exercise, and low nutritional availability will be discussed as factors that up regulate the enzymatic activity of SIRT1 in various tissues.

Aging

The process of aging is a matter of much interest in the field of science. Many diseases are the result of aging, hence learning as much as possible about this process is imperative in order to understand how to slow down, prevent, or even avoid aging-related diseases. There are many theories about how the aging process occurs, but this review will focus on the theory that is indirectly sustained by the studies presented. The Free Radical Theory of Harman proposes that aging occurs when macromolecules and cells become oxidized

(LaGuire and Reaves 2013). Chemists have classified reactions in numerous ways such as combustion, acid-base, displacement, neutralization, and redox reactions. Redox reactions involve the transfer of electrons in which the particle that gains an electron is the reduced particle and the one that loses an electron is the oxidized particle. Under normal conditions, the inside of cells is maintained reduced through a constant input of metabolic energy. This means that the inside of the cell has many agents that tend to give away their electron, thus reducing any unstable molecule, like free radicals. Oxidative stress happens when the production of reactive oxygen surpasses the ability of the biological system (whether it may be a cell, mitochondrion or macromolecule) to detoxify the intermediates produced, called free radicals or reactive oxygen species (ROS), resulting in damage (LaGuire and Reaves 2013). The damage of cellular function correlates with a short lifespan, not only of the cells but the body as a whole.

The activity of SIRT1 diminished considerably in lung cells treated in a dose and time dependent manner with cigarette smoke extract and hydrogen peroxide (Caito et al. 2010). It was also found that under these conditions SIRT1 leaves the cell nucleus, thereby failing to regulate targeted genes and proteins. Since cigarette smoke contains many chemicals that cause inflammation of the lung tissue, this study serves to explain how inflammation and aging (represented by the time duration of the treatments) cause chronic oxidative stress, thus impairing the SIRT1 deacetylase activity, rendering it inactive, and shortening the lifespan. (Caito et al. 2010)

Settling the debate: Hypothalamic SIRT1 as key to longevity Page 3

Review Article 2013

Exercise

Exercise has been identified as a mechanism to increase the activity of SIRT1 in human skeletal muscle (Gurd et al. 2010). The study carried out by Gurd and his colleagues stands out because it involved human subjects. Samples of skeletal muscle were taken from the human subjects through biopsies before and after the study in order to have a control group that could serve for comparison. After a 6-week training period samples were analyzed. The activity of SIRT1 protein was found to have increased c with the increase correlating with the muscle’s oxidative capacity. On the other hand, the protein count had decreased. This is a novel finding that contradicted expectations (Gurd et al. 2010).

An explanation for this could be that oxidative stress activates SIRT1 to start the process of deacetylating FOX03 avoiding cell death by responding to the increase of activity. But since this study was based on high-intensity exercise, it could be assumed that the oxidative stress in muscle cell was starting to cause the SIRT1 to leave the nucleus, and possibly being damaged as a macromolecule by the free radicals. Keep in mind that SIRT1 proteins are enzymes and therefore have access to a greater increase in substrate activity. The SIRT1 proteins left in the nucleus might be less, but have an increase in activity in order to deacetylate as much FOX03 as possible to counterbalance the effects of oxidative stress.

Caloric Restriction

One of the most common strategies used in studies of life span extension is caloric

restriction (CR). The technique implies lowering caloric intake below the levels for maximum development and fertility but still managing to be considered nutritionally sufficient. Many studies have shown that CR has a stress effect in the body. In turn, this stress activates the body’s survival mechanisms. The body will always strive to regain homeostasis in order to function properly. SIRT1 is an important key component of the regulation of cells and their adaptations to stress. In other words, since CR produces a stressful situation for cell, it is very likely that SIRT1 is involved in this process as the main regulator to counterbalance the effect of CR. This possibility motivated many researchers to conduct even more studies.

When food intake is restricted, the body’s metabolism generates less glucose in response to the low nutritional availability. Since there is less glucose, the mitochondria of cells will produce low quantities of adenosine triphosphate (ATP, the most common energy provider for cells), which correlates with an increase of NAD+, an important component that is reduced to NADH in the citric acid cycle of cellular respiration. SIRT1, like other Sirtuin proteins of the family, is NAD-dependent. A higher content of NAD+ causes an increase in SIRT1 deacetylase activity. When SIRT1 is activated, it deacetylases its target substrates like PGC-1a and FOX proteins. They are responsible for the expression of genes that regulate mitochondrial biogenesis and activity. If there is an ongoing healthy mitochondrial activity, then there will be an improved metabolism and disease prevention (Houtkooper et al. 2012). On the contrary, caloric excess helps the body produce even more glucose, which

Settling the debate: Hypothalamic SIRT1 as key to longevity Page 4

Review Article 2013

activates the citric acid cycle of mitochondria. More ATP will be produced and, consequently, more NAD+ will be reduced to NADH. Low NAD+ levels inhibit the activity of SIRT1, causing PGC-1a to remain acetylated and mitochondrial activity to decrease (Houtkooper et al. 2012). This has been related to predisposition of developing metabolic diseases.

From the studies that have helped shaped this relationship between caloric intake and SIRT1 it would appear that caloric restriction is recommended if one wants to be healthy and live longer. It is important to clarify that the studies supporting this general hypothesis were focused mainly in the expression and activity of SIRT1 in tissues like skeletal muscle, kidney, and liver. Nonetheless, Ҫakir and colleagues (2009) provide intriguing insight contrary to this hypothesis by analyzing hypothalamic SIRT1. According to Ҫakir’s study, caloric restriction, instead of extending life span, actually shortens it by causing obesity and related diseases. Hypothalamic SIRT1 protein content and activity increased as NAD+ levels grew. Specifically, this was noted in the ARC and PVN nuclei of the hypothalamus. SIRT1 was found to deacetylate FOX01 in these parts of the hypothalamus, which in turn, regulates the transcriptional activity in the thyroid hormone (Singh et al. 2013). Both PGC-1a (Amat et al. 2009) and FOX01 (Singh et al. 2013) substrates promote gluconeogenesis. Studies concluded that SIRT1 promotes longevity by means of gluneogenesis, a conclusion shared with other studies that have focused on the role of SIRT1 in other tissues of the body. However, Ҫakir’s study went a little further. The novel finding of this study stated that the deacetylation of

FOX01 suppressed the expression proopiomelanocortin (POMC), which is the precursor molecule of a-MSH, an appetite regulator. This decrease in POMC expression correlated with an increase in agouti-related peptide (AgRP) stimulating appetite and food intake, resulting in weight gain, and ultimately obesity (Ҫakir et al. 2009).

The appearance of contradictory evidence like this is what has enhanced the intense debate of the role of SIRT1 protein in longevity. Nonetheless, a very recent study has managed to successfully link SIRT1 to longevity and provide enough evidence to clarify the contradictory results of other experiments and finally settle the debate. The study conducted by Satoh and his colleagues (2013) has recently given substantial proof that protein Sirtuin 1 (Sirt1) plays an important role in longevity and the process of aging. In this new study, transgenic BRASTO mice overexpressing Sirt1 specifically in the dorsomedial and lateral hypothalamic nuclei were examined under diet restriction conditions. According to the results, Sirt1 works by deacetylating protein Nkx2-1, thus enhancing the latter’s transcriptional activity and the expression of its target gene Ox2r. In turn, the target gene enhances neural activation in the DMH and LH nuclei, stimulating the sympathetic nervous system and ultimately skeletal muscle function (Satoh et al. 2013). Also, this enhanced neural activity preserves the quality of sleep. Having functional muscles mean that muscles have the capacity to metabolize and use energy, which is exactly how mitochondrial biogenesis works. Other neurobehavioral responses were an increase of physical activity, body temperature and oxygen consumption.

Settling the debate: Hypothalamic SIRT1 as key to longevity Page 5

Review Article 2013

Altogether, these responses protect skeletal muscle against age-related declines and diseases by maintaining a youthful appearance overall (Satoh et al. 2013). The most important component of this study is that they compared two different lines of BRASTO mice. Line 1 (studied in a previous investigation in 2010), overexpressed SIRT1 throughout all the hypothalamic nuclei, whereas line 10 only overexpressed the protein in the DMH and LH nuclei. The group of investigators noted that the BRASTO mice form line 1 had a higher neural activity during dark time when compared to controls; there was no enhancement of neural activity, which contrasted to the high neural activity in the ARC nuclei. From this observation, it was concluded the activation of neural activity on the ARC suppresses the neural activation in DMH and LH resulting in no delay of aging and ultimately, no life span extension (Satoh et al. 2013). These results could explain why the overexpression or hyperactivation of SIRT1 in other areas of the body do not extend life span.

Conclusion

SIRT1 has been the center of an intense debate since the first moment it was identified and related to a possible life span extension and longevity. The different results that have risen from various studies have fomented this debate. SIRT1 cannot be linked to such a complex term like longevity based on one or two studies. However, after analyzing various studies, it is clear that the reason for the dispute lies in the different techniques and approaches used to analyze and characterize the role of

SIRT in longevity. Satoh’s study is a breakthrough because it proposes a novel pathway that connects SIRT1 not to survival and homeostasis like other studies have done, but to genuine longevity and healthy body function. The neurobehavioral responses that BRASTO mice exhibited like better sleep quality, muscle function and morphology are all characteristics of a healthy and young organism. We will achieve longevity as well as we achieve these qualities through time. Science is heading towards understanding how aging works in order to find ways to slow down the process, maybe even prevent or avoid aging-related diseases. With each new study and investigation, SIRT1 is proving to be the key to longevity. Nonetheless, more research is needed in order to formally establish this relationship.

References:

1. Amat R, Planavila A, Chen SL, Iglesias R, Giralt M, Villarroya F. 2009. SIRT1 controls the transcription of the peroxisome proliferator-activated receptor-gamma Co-activator-1alpha (PGC-1alpha) gene in skeletal muscle through the PGC-1alpha autoregulatory loop and interaction with MyoD. J Biol Chemistry [Internet; cited 2013 Oct 17] Doi:10.1074/jbc.M109.022749 [284(33): 21872–21880] Available in: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755911/

2. Caito S, Rajendrasozhan S, Cook S, Chung S, Yao H, Friedman AE, Brookes PS, Rahman I. 2010. SIRT1

Settling the debate: Hypothalamic SIRT1 as key to longevity Page 6

Review Article 2013

is a redox-sensitive deacetylase that is post-transcriptionally modified by oxidants and carbonyl stress. FASEB J. 24(9): 3145-3159

3. Ҫakir I, Perello M, Lansari O, Messier NJ, Vaslet CA, Nillni EA. 2009. Hypothalamic SIRT1 Regulates Food Intake in a Rodent Model System. PLoS ONE. 4(12): e8322. doi: 10.1371/journal.pone.0008322.

4. Gurd BJ, Perry CGR, Heigenhauser GJF, Spriet LL, Bonen A. 2010. High-intensity interval trining increases SIRT1 activity in human skeletal muscle. Appl. Physiol. Nutr. Metab. 35: 350-357

5. Houtkooper RH, Pirinen E, Auwerx J. 2012. Sirtuins as regulators of metabolism and healthspan. Nature Reviews. Molec. Cell Biol. 13(4): 225-238

6. Kelly, G. 2010. A Review of the Sirtuin System, its Clinical Implications, and the Potential Role of Dietary Activators like Resveratrol: Part 1. AMR. 15(3): 245-263

7. LaGuire TC, Reaves SK. 2013. The Sirtuins in Aging and Metabolic Regualtion. Sci. Res. 4: 668-677

8. Reece JB, Urry LA, Cain ML, Wasserman SA, Minorsky PV, Jackson RB. 201. Campbell Biology. 9th Edition. San Francisco, CA: Pearson.Glycolisis and the citric acid cycle connect to many other metabolic pathways: The Versatility

of Catabolism. Figure 9.19 The catabolism of various molecules from food; p.180.

9. Satoh A, Brace CS, Rensing N, Cliften P, Wozniak DF, Herzog ED, Yamada KA, Imai S. 2013. Sirt1 Extends Life Span and Delays Aging in Mice through the Regulation of Nk2 Homeobox 1 in the DMH and LH. Cell Metabolism. 18: 416-430.

10. Singh BK, Sinha RA, Zhou J, Xie SY, You SH, Gauthier K, Yen PM. 2013. FOX01- Deacetylation regulates Thyroid Hormone Induced Transcription of Key Hepatic Gluconeogenic Genes. J Biol Chemistry. [Internet; cited 2013 Nov 7] Doi: 10.1074/jbc.M113.504845 [288: 30365-30372] Available in: http://www.jbc.org/content/early/2013/08/30/jbc.M113.504845.full.pdf+html

Additional References

1. ArGP Medical Definition Search [Internet] Medilexicon [cited 2013 Nov 9] Available at: http://www.medilexicon.com/medicaldictionary.php?s=agouti-related+peptide

2. General aspects of mitochondrial biogenesis [Internet] Mitochondrial biogenesis [cited 2013 Nov 7] Available at: http://commonweb.unifr.ch/biol/pub/zoology/Homepage/Trypanos/projects/mitogeneralities.htm

3. Health Concerns. Caloric restriction [Internet] Life Extension Foundation

Settling the debate: Hypothalamic SIRT1 as key to longevity Page 7

Review Article 2013

for Longer Life [cited 2013 Nov 9] Available at: http://www.lef.org/protocols/lifestyle_longevity/caloric_restriction_01.htm

4. Online Resources for Disorders Caused by Oxidative Stress [Internet] Online Resources for Disorders Caused by Oxidative Stress [cited 2013 Nov 7] Available at: http://www.oxidativestressresource.org/

5. POMC Medical Definition Search [Internet] Medilexicon [cited 2013 Nov 9] Available at: http://www.medilexicon.com/medicaldictionary.php?s=proopiomelanocortin

Settling the debate: Hypothalamic SIRT1 as key to longevity Page 8