Does Sirtuin protein family Sir2 work in low-calorie diet mostly?

Does Sirtuin protein family Sir2 work in low-calorie diet mostly?

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I am reading about the protein family in relation to the prolongation of cell life. It is known that

Sirtuins have been implicated in influencing a wide range of cellular processes like ageing, transcription, apoptosis, inflammation and stress resistance, as well as energy efficiency and alertness during low-calorie situations.

However, I am not sure if these physiologic processes are only during low-calorie situations. Alertness is at least.

It says in the original paper only that

Our results suggest that in the hypothalamus, SIRT1 functions as a key mediator of the central response to low nutritional availability, providing insight into the role of the hypothalamus in the regulation of metabolism and aging in mammals.

However, it does not specify how the Sir2 works to high-calorie diet. Therefore, I asked mostly. When is the protein family active?

Does the Sirtuin protein family work mostly during low-calorie diet?

Does sirtuin 1 (SIRT1/SIR1) function to mediate the effects of a low calorie diet? Perhaps, but its really not clear. Is the function of SIRT1 primarily to mediate low calorie response? Absolutely not. What does that mean for SIRT2/SIR2 - its probably not more closely associated with calorie restriction signals, though it might be connected in a similar ambiguous way… but see below.

Firstly SIRT1 has also been shown to have many influences in many studies of conditions such as Alzheimers, anxiety and other neuro-functions. SIRT1 modifies p53 - a bottleneck in cellular reproduction and a key regulator in cancer prevention, but which also an important aspect as to why it is active in calorie restriction conditions.

When reading reference like the one you have here its important to remember that correlation is not causality. Very few genes in animals function only when specific behavior or conditions are experienced, especially signaling genes.

Examining the OMIM report (see link above) there has been a close examination of the possible role of SIRT1 in calorie restriction since the early 2000s in knockout mice.

SIRT1 would seem to play an important role in embryonic development for instance - cell growth is so important at that point and 80% of mice who are homozygous SIRT1 (-/-) knockouts die before they are born and even more die in the early natal period. The mice who do survive have problems generating red blood cells which is another important case of cellular reproduction.

Yet the relationship of the knockout mice to calorie restriction has been debated - its been difficult to interpret the activity and the weight loss or gain of the knockout mice compared to wild type mice since the SIRT1 knockouts are smaller. They may also have genetic traits that compensate for the absence SIRT1 in order to have survived.

More recently mice where SIRT1 expression in the brain have been produced, but the result has been anxiety or anxiousness as opposed to specific calorie restriction diet.

This reminds me of the story of leptin, in the early 90s they found that leptin deficient mice became quite fat, but despite this obvious linkage to obesity, leptin turned out to mediate a lot of other psychological factors as well - it only caused obesity when its function was blocked. It did not turn out to be an appetite signal.

SIRT2 seems to have similar roles in calorie restriction.

Rogina et al (2002) found that under 2 life-extending conditions, Rpd3 (601241) mutants fed normal food and wildtype flies fed low calorie food, Sir2 expression was increased 2-fold.

But also has similar relationships to Alzheimers. SIRT2 has not been followed up on as much as SIRT1. This correlation might not be causal or should be nuanced in a similar way.

Does Sirtuin protein family Sir2 work in low-calorie diet mostly? - Biology

Silent information regulator 2 (Sir2) proteins, or sirtuins, are protein deacetylases/mono-ADP-ribosyltransferases found in organisms ranging from bacteria to humans. Their dependence on nicotinamide adenine dinucleotide (NAD + ) links their activity to cellular metabolic status. In bacteria, the sirtuin CobB regulates the metabolic enzyme acetyl-coenzyme A (acetyl-CoA) synthetase. The earliest function of sirtuins therefore may have been regulation of cellular metabolism in response to nutrient availability. Recent findings support the idea that sirtuins play a pivotal role in metabolic control in higher organisms, including mammals. This review surveys evidence for an emerging role of sirtuins as regulators of metabolism in mammals.

Present address: Howard Hughes Medical Institute, Children's Hospital Boston, Immune Disease Institute, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

Key Points

Sirtuins link NAD + cleavage to deacetylation of target proteins that are important in metabolism

SIRT1 regulates key factors that influence hepatic carbohydrate and lipid metabolism

SIRT1 counteracts processes that result in cardiovascular disease and the metabolic syndrome

SIRT3 regulates oxidative metabolism and suppresses the production and the effects of reactive oxygen species

SIRT6 regulates glucose homeostasis

Sirtuins could be pharmacologically activated to protect against metabolic and age-related diseases

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Role of Sirtuins in Cardiac Hypertrophy

In general, cardiac hypertrophy is the primary response of the heart to hemodynamic overload, such as pressure or volume overload, and is characterized by enhanced protein synthesis and sarcomeric reorganization, leading to increased cardiomyocyte size. Hypertrophic growth initially reduces wall stress, maintains cardiac function, and serves as a compensatory response to hypertrophic stimuli, but it eventually initiates the development of cardiac remodeling and heart failure. Pathological hypertrophy is recognized as a strong independent factor in heart failure. Reversible modification of the acetylation status of proteins by histone acetyltransferases and HDACs critically regulates the activity of signaling molecules mediating cardiac hypertrophy (34). Class I HDACs, including HDAC1, 2, 3, and 8, positively regulate cardiac hypertrophy, whereas class II HDACs, including HDAC4, 5, 7, and 9, negatively regulate cardiac hypertrophy (152). Class II HDACs are expressed only in nonproliferative cells, including cardiomyocytes. The export of class II HDACs from the nucleus to the cytosol in the heart directly induces transcription of nuclear factor of activated T cell and MEF2, master positive regulators of cardiac hypertrophy (9). Class II HDACs are regulated by several forms of post-translational modification, including phosphorylation, ubiquitination, and sumoylation (9, 52, 108). Recently, it was shown that Trx1 regulates the nucleocytoplasmic shuttling of HDAC4 through a redox-dependent mechanism (3). By forming a multiprotein complex with Trx1-binding protein-2 and DnaJb5, Trx1 reduces HDAC4 at C667 and C669, which are easily oxidized to form a disulfide bond in response to hypertrophic stimuli. The redox status of these cysteines plays a critical role in the localization of HDAC4, thereby regulating cardiac hypertrophy (3).

Sirtuins have been demonstrated to be involved in cardiac hypertrophy, although their effects vary depending upon experimental conditions. Resveratrol attenuates the phenylephrine-induced hypertrophic response in cardiomyocytes and pressure overload-induced cardiac hypertrophy. Pharmacological inhibition of Sirt1 (with NAM or sirtinol) decreases, whereas overexpression of Sirt1 increases the size of cardiomyocytes (6). Overexpression of Sirt1 attenuates endothelin-1-induced cardiomyocyte hypertrophy in vitro via degradation of H2A.z. Upregulation and activation of Sirt1 induced by phenylephrine (an α-adrenergic agonist) are blocked by inhibition or downregulation of AMPK, leading to attenuated cardiac hypertrophy. Phenylephrine-induced hypertrophy in cardiomyocytes is attenuated by resveratrol and overexpression of Sirt1. This antihypertrophic effect is canceled by downregulation of PPAR-α. On the other hand, pressure overload coupregulates Sirt1 and PPAR-α in the heart, which coordinately suppress genes that are regulated by ERRs, thereby leading to the development of cardiac hypertrophy and heart failure (Fig. 1) (97). Importantly, haploinsufficiency of PPAR-α and Sirt1 attenuates cardiac hypertrophy in response to pressure overload (97). Interestingly, although low (2.5-fold) to moderate (7.5-fold) levels of overexpression of Sirt1 in the heart attenuate age-dependent cardiac hypertrophy, a high level (12.5-fold) of Sirt1 increases it (5). Thus the effect of Sirt1 upon cardiac hypertrophy may be affected by a balance of interaction with other factors, such as PPAR-α, or severity and type of stress. In addition, cardiac hypertrophy is a multifactorial disease and may also be affected by angiogenesis, apoptosis, and fibrosis. Akt is one of the key molecules regulating cardiac hypertrophy. A recent study demonstrated that Sirt1 critically regulates Akt activation by deacetylation at lysine residues in the pleckstrin homology domains of Akt. Acetylation of Akt and phosphoinositide-dependent protein kinase 1 (PDK1) blocks binding of Akt and PDK1 to PIP3. Deacetylation of Akt by Sirt1 enhances binding of Akt and PDK1 to PIP3 and promotes their activation, thereby leading to cardiac hypertrophy (Fig. 1) (125). On the other hand, Sirt1 knockout mice have smaller hearts than wild-type mice and exhibit resistance to the development of cardiac hypertrophy in response to hypertrophic stimuli. Overall, these findings lead to speculation that the role of Sirt1 as a progrowth or antigrowth factor for cardiomyocytes is tightly controlled by the magnitude of Sirt1 expression.

Sirt3 has also been shown to confer resistance to cardiac hypertrophy. Sirt3 is upregulated during mild cardiac hypertrophy, whereas it is decreased in severe cardiac hypertrophy (126). Overexpression of Sirt3 causes suppression of agonist-induced cardiac hypertrophy. Conversely, Sirt3 knockout mice exhibit exacerbation of the hypertrophic response. The beneficial effects of Sirt3 are mediated by the activation of FoxO3-dependent transcription of catalase and MnSOD, suppression of ROS-induced Ras activity, MAPK/ERF, and Akt/PI3K signaling (Fig. 2) (124). Exogenous NAD + treatment blocks antihypertrophic liver kinase B1-AMPK signaling, which is accompanied by activation of Sirt3 but not Sirt1(103), indicating the critical role of Sirt3 in cardiac hypertrophy and redundancy in Sirt1 targets.

Fig. 2.Signaling pathways of Sirt3 in the heart. Sirt3 regulates Mitochondrial permeability transition pore (mPTP) opening, apoptosis, and cell survival, mainly through deacetylation of mitochondrial proteins, including cyclophilin D (CypD), Ku70, and complex I, independently of transcription. Sirt3 also deacetylates FoxO, leading to transcriptional upregulation of MnSOD and catalase and inhibition of cardiac hypertrophy.

The role of Sirt6 in cardiac hypertrophy has been investigated using loss- and gain-of-function models (127). Sirt6 knockout mice exhibited cardiac hypertrophy and heart failure, whereas overexpression of Sirt6 attenuated cardiac hypertrophy, indicating that Sirt6 negatively regulates cardiac hypertrophy. Unlike Sirt1 and Sirt3, Sirt6 functions to directly attenuate insulin-like growth factor (IGF)-Akt signaling at the chromatin level (Fig. 3) (127).

Fig. 3.Signaling pathways of Sirt6 in the heart. Sirt6 deacetylates histones at Lys9, thereby suppressing the insulin-like growth factor (IGF)-Akt pathway and cardiac hypertrophy. Sirt6 increases the resistance to hypoxic injury in the heart by stimulating AMPK-α and Bcl2 and inhibiting NK-κB and Akt. pAkt, phospho-Akt Ac, acetylated.

Whereas a recent study demonstrated that Sirt2 is required for optimal Akt activation in insulin- and growth factor-responsive cells, the role of Sirt2 in cardiac hypertrophy is still unclear. Likewise, although Sirt4, Sirt5, and Sirt7, like Sirt1 and Sirt3, appear to be protective against stress in the heart, there has been no report thus far regarding the role of these proteins in cardiac hypertrophy.


Sirtuins has generated considerable interest as an important player in aging biology. It is generally agreed that sirtuins have a role in expanding the lifespan in laboratory model organisms by mediating the anti-aging effects of a low-calorie diet (calorie restriction) (Haigis & Guarente, 2006 ). In mammals, there are seven sirtuin homologues (SIRT1 to SIRT7), which possibly have a role in adapting to food deprivation and other environmental stressors. Of these seven, the biological activities of SIRT1 and SIRT2 have been well studied. SIRT1 is a nuclear protein in most cell types. It deacetylates transcription factors and cofactors that regulate several metabolic pathways in energy metabolism (Li et al., 2007 ) and circadian rhythm (Nakahata et al., 2008 ). SIRT1 is believed to have important function in glucose homeostasis and lipid metabolism in various tissues including adipose tissues, liver, pancreas, and skeletal muscle (Kume et al., 2010 ). Other SIRT1 targets are related to stress tolerance, DNA repair, (Nakagawa & Guarente, 2011 ), and inflammation (Yeung et al., 2004 ). Sir2 mediates transcriptional silencing at selected regions of the yeast genome, and expanded lifespan of yeast mother cells has been shown to be associated with increased the silencing activity of Sir2 at the ribosomal DNA repeats (Kaeberlein et al., 1999 ).

Several reports on association between sirtuins and disease conditions such as diabetes and metabolic diseases cardiovascular diseases neurodegenerative diseases and cancers have been published in recent years mostly in animal models (Guarente, 2014 ). Incidentally, all these conditions are associated with aging process. There is substantial evidence to relate sirtuins to the aging process, age-related diseases, and anti-aging interventions. In the present study, an attempt was made to relate sirtuins to a major geriatric syndrome that is frailty. Serum sirtuins (SIRT1, 2 and 3) were significantly lower in frails as compared to nonfrail after adjustment for multiple confounders such as age, gender, diabetes mellitus, hypertension, cognitive impairment, and number of comorbidities. SIRT 1 and 3 levels decreased in patients with diabetes and hypertension, but the levels were even lower in individuals who were frail and had diabetes or hypertension. SIRT 1 and 3 levels also fell as age and the number of comorbidities increased, but the decline was more in frailer subjects compared with nonfrail elderly. The level of SIRT 2 was also lower in frail compared with nonfrail reaching the level of statistical significance.

This the first report associating significantly low circulating sirtuin with frailty. The results of SPR technology was validated by Western blot analysis considered as gold standard for the analysis of proteins. Earlier studies conducted on CHAMP population assessed the SIRT1 expression in SK Hep1 cells grown in the presence of serum samples obtained from frail or nonfrail individuals and found there was no association between frailty and SIRT1 expression in cells. The post hoc analysis suggested that there might be a paradoxical association between low serum-induced SIRT1 expression and robustness (Le Couteur et al., 2011 ). Moreover, the authors of the CHAMP study accepted that their results were unexpected, as high tissue expression of SIRT1 was generally considered to be beneficial, it was induced by CR and expected to be higher in younger animals.

There are some biological parameters which have been used as biomarkers of frailty, namely inflammation (Leng et al., 2002 , 2004 , 2007 , 2009 ), central adiposity, serum albumin, oxidative stress (Wu et al., 2009), vitamin-E (Ble et al., 2006 ), and 25 hydrooxy vitamin-D level (Hirani et al., 2013 ). These parameters are, however, nonspecific and do not indicate any mechanistic pathway. Despite rise in the number of frail patients in clinical practice, so far, no diagnostic biomarker has been identified for frailty, possibly due to decline in multiple physiological functions. Better understanding of the anti-aging effects of CR at a molecular level may ultimately help in the development of potent diagnostic and therapeutic target for frailty, and sirtuins may emerge as key molecules in this regard.

ROC results showed that serum SIRT1 and SIRT3 have great potential to be diagnostic protein marker for frailty for its accuracy in the study cohort. This is the first study to report the clinically diagnostic relevance of SIRT1 and SIRT3 as serum protein marker for frailty.

Cellular metabolism underlying effects of IF

In addition to its role as an energy carrier, beta-hydroxybutyrate (βOHB) binds to extracellular receptors and inhibits class I histone deacetylases, which may promote resistance to oxidative stress [57,58]. Thus, epigenetic modifications are likely driven by fasting-induced βOHB elevations. Increases in βOHB alter gene expression in nutrient-sensitive pathways that are implicated in longevity. βOHB also has been shown to promote anti-inflammatory effects by blocking activation of the NLRP3 inflammasome [59] and activating a neuroprotective subset of macrophages in the mouse brain [60]. In addition to the association between IF and neuronal resistance, animal studies have found that IF can affect oxidative stress.

Oxidative stress

CR reduces oxidative stress by limiting mitochondrial generation of ROS and increasing endogenous antioxidant activity this results in reduced oxidative damage to cellular proteins, lipids, and nucleic acids [61,62]. However, IF has mixed effects on oxidative stress in animal models [63]. In theory, IF may induce hormesis, resulting in beneficial adaptive changes that include activation of AMP-activated protein kinase, mitochondrial network and peroxisome remodeling, and increased production of antioxidant enzymes [42,64,65].

In 8 month old mice at high risk of lymphoma, ADF was associated with a significant reduction in lymphoma (0% vs. 33% of controls), decreased spleen mitochondrial ROS generation, and increased antioxidant superoxide dismutase activity [66]. However, Cerqueria reported increased oxidative stress in 8-week-old Sprague-Dawley rats who underwent 32 weeks of ADF [67]. The ADF group had worsened glucose tolerance, lowered adiponectin, and increased insulin receptor nitration and release of ROS in intra-abdominal AT and muscle [67]. Hence unlike long-term CR, long-term ADF may be associated with worsened Si and oxidative stress. Another study further informed these mixed results. One month of ADF in 8-week old Sprague-Dawley rats had complex, tissue-specific effects on ROS balance in rats [68]. For example, biomarkers of oxidative damage were increased in the liver and the brain but were reduced in the heart [68]. Hence, the effects of IF depend on the animal model, age at initiation, and tissue sampled.

Autophagy is a catabolic process of nutrient recycling that is essential for defense against oxidative stress. Nutrient sensing pathways induce autophagy [69]. IF has been shown to restore autophagic function, thereby preserving organelle quality. This restorative function is impaired by insulin resistance [70] and obesity-induced diabetes in mice fed a high fat diet [71]. However, data on the mechanisms of IF in humans are limited. Twenty-three pre-menopausal women (BMI 25�.9 kg/m 2 ) followed an ICR diet (two days per week of 65% CR) for one menstrual cycle [72]. After the intervention, 196 metabolites increased (including βOHB and acylcarnitine) and 331 metabolites significantly decreased (including succinic acid, alanine, glutamic acid, and tyrosine). This group also compared the effects of their ICR protocol on the metabolome in another group of pre-menopausal women and found many similar trends [73].

Inflammatory effects

Oxidative stress is closely linked to inflammation. Ten subjects with obesity and asthma followed an ADF protocol for 2 months [36]. Body weight decreased a mean 8% and peak expiratory flow and asthma quality of life scores increased [36]. This intervention was associated with significant reductions in inflammatory markers, including TNF-α and ceramides, and markers of oxidative stress, including protein carbonyls and 8-isoprostane.

In mice, IF increases vascular endothelial growth factor (VEGF) in WAT, with associated alternative macrophage activation and WAT beiging [74]. Gene expression of VEGF, alternative macrophage activation, and beige adipocyte-related proteins are also positively correlated in human AT. This suggests that IF may regulate this same pathway.

Fasting is associated with elevations in FFA and ketone bodies, including βOHB, which may have opposing effects on inflammation. Elevated FFA during fasting may activate proinflammatory pathways [75] and reduce Si [76], while elevations in βOHB may activate anti-inflammatory pathways and alter fuel metabolism as reviewed above. Our group is currently conducting a pilot clinical trial on the effects of dietary supplementation of medium chain triglycerides (MCT), which are metabolized into βOHB ( <"type":"clinical-trial","attrs":<"text":"NCT02783703","term_id":"NCT02783703">> NCT02783703). MCT supplementation may activate anti-inflammatory pathways through βOHB without the detriments of elevated FFAs.

FFA released from lipolysis in mast cells may play an important role in eicosanoid release and control of immune activation [77]. Saturated FFA induce an inflammatory response in macrophages while unsaturated FFA do not [78]. Hence, FFA released from lipolysis play an important role in obesity-induced AT inflammation [79], immune regulation [80], and stimulating hepatic VLDL production [81].

Sustained fasting is associated with acute hepatic steatosis and increased insulin resistance [82]. Normal weight subjects who fasted for 72 and 120 hours had increased intramuscular lipids (IMCL) [83,84]. A 60-hour fast in healthy males was associated with elevated IMCL, increased insulin resistance, and a nine-fold elevation in FFA [83]. Prolonged elevations in FFA, combined with metabolic syndrome and insulin resistance, contribute to increasing hepatic IMCL and lipotoxicity, which leads to nonalcoholic steatohepatitis [84]. However, ADF in mice has been shown to induce metabolic changes that protect against steatosis [85]. Increases in ketogenesis during fasting protects against steatohepatitis in mice [86] thus, IF may have these same protective effects in humans.

The effects of IF on inflammation have been minimally studied in humans. While cellular level mechanisms have been evaluated in animal models, the application in human models is scarce. Cellular analysis and animal models suggest opposing influence of FFA and ketone bodies on inflammation. Despite understanding these cellular mechanisms, it is unclear whether IF has beneficial effects on oxidative stress and inflammation in human.

Conclusions and future directions

Diet, exercise and other aspects of our daily interaction with the environment have the potential to alter our brain health and mental function. We now know that particular nutrients influence cognition by acting on molecular systems or cellular processes that are vital for maintaining cognitive function. This raises the exciting possibility that dietary manipulations are a viable strategy for enhancing cognitive abilities and protecting the brain from damage, promoting repair and counteracting the effects of aging. Emerging research indicates that the effects of diet on the brain are integrated with the actions of other lifestyle modalities, such as exercise (see BOX 2) and sleep 131 , 132 . The combined action of particular diets and exercise on the activation of molecular systems that are involved in synaptic plasticity has strong implications for public health and the design of therapeutic interventions. Owing to the encouraging results of clinical and preclinical studies that showed the beneficial effects of foods on the brain, the topic has attracted substantial media attention. Some of the information that has been conveyed has been hazy or exaggerated, and has contributed to people’s apprehension of taking advantage of scientific advances. As discussed, several dietary components have been found to have positive effects on cognition however, caution is required, as a balanced diet is still the stepping-stone for any dietary supplementation. By the same token, popular dietary prescriptions that might help to reduce weight do not necessarily benefit the physiology of the body or the mind.

Brain networks that are associated with the control of feeding are intimately associated with those that are involved in processing emotions, reward and cognition. A better understanding of how these networks interact will probably produce fundamental information for the development of strategies to reduce food addiction and obesity, a major social and economic burden in Western society. It is encouraging that modern psychiatry has started to appraise the implementation of some of these concepts for the treatment of various mental disorders. For example, a consensus report from the American Psychiatric Association’s Committee on Research on Psychiatric Treatments has provided general guiding principles for the use of omega-3 fatty acids for the treatment of mood disorders 80 .

The fact that dietary factors and other aspects of lifestyle have an effect on a long-term timescale contributes to an under-estimation of their importance for public health. Accordingly, the slow and imperceptible cognitive decay that characterizes normal aging is within the range-of-action of brain foods, such that successful aging is an achievable goal for dietary therapies. The capacity of diet to modulate cognitive abilities might have even longer-term implications in light of recent studies that imply that nutritional effects might be transmitted over generations by influencing epigenetic events. Research indicating that an excessive intake of calories might negate the positive effects of certain diets suggests that there is an undefined line between abundance of foods and neural health. Ironically, judging by the increasing rate of obesity in Western countries, which affects individual’s health and the economy as a whole, the excessive food intake in these wealthy nations seems to be almost as harmful as the lack of it in poor countries. It is intriguing that several countries with limited resources, such as India, have a reduced prevalence of neurological disorders that have been associated with diet, such as Alzheimer’s disease. This raises the concern of whether industrialized societies are consuming a balanced diet that takes into consideration appropriate numbers of calories as well as appropriate nutrients and adequate levels of exercise. Many practical questions regarding the design of diets to specifically improve brain function, such as type, frequency and amount of nutrients that constitute healthy brain food, remain to be answered, but we are beginning to uncover the basic principles that are involved in the actions of foods on the brain. Incorporating this knowledge into the design of novel treatments could be vital to combating mental diseases and neurological weaknesses.

Modulators of epigenetic pathways

The term epigenetics denotes heritable phenotypic alterations caused by postreplicative modifications of chromatin, rather than classical mutation-based genetic changes. Such covalent and noncovalent modifications of DNA and proteins (e.g., histones) alter the state of chromatin conformation and elicit corresponding changes in transcriptional activity (Jaenisch & Bird, 2003 Goldberg etਊl., 2007 Baker etਊl., 2008). Epigenetic effects can be elicited by three distinct principal means: (i) DNA methylation (ii) post-translational histone modifications and (iii) noncoding RNA interference (Goldberg etਊl., 2007 Baker etਊl., 2008). Twin studies suggest that genetics at birth determines only 25% of lifespan therefore, it is proposed that epigenetic factors also contribute to aging. Such epigenetic factors are likely influenced by lifestyle, diet, and exogenous stress, raising the possibility that strategies can be developed to ameliorate age-associated cellular dysfunction (Imai etਊl., 2000 Longo, 2009).

Although manipulation of enzymes (sirtuins, histone acetyltransferases, histone deacetylases) that regulate the (de)acetylation status of chromatin (and other targets) can prolong lifespan in yeast, flies, and worms, the role of histone modifications in lifespan regulation is poorly understood. A fly model has recently been introduced in which the impact of such histone mutations on aging and lifespan can be evaluated (Pengelly etਊl., 2013).

A naturally occurring polyamine, spermidine, directly inhibits histone acetyltransferases (HATs), thereby maintaining histone H3 in a hypoacetylated state (Eisenberg etਊl., 2009). Functionally, this results in higher resistance to heat and oxidative stress as well as markedly reduced rates of cell necrosis during aging in human and yeast cells. Strikingly, this mechanism extends chronological lifespan across species, including flies, nematodes, and human cells. These data support the existing body of knowledge regarding histone acetylation in lifespan maintenance, including the finding that deletion of sas2, encoding a histone acetyltransferase, extends the replicative lifespan in yeast (Dang etਊl., 2009). Sas2 antagonizes Sir2, a prominent histone deacetylase involved in aging, and its deletion stabilizes Sir2 levels in aging cells, thereby allowing a low basal level of acetylation on specific histone residues associated with longevity regulation (Raisner & Madhani, 2008). Finally, a simple way to change age-related histone acetylation consists of dietary strategies that deplete cellular acetyl CoA, the sole donor for acetylation reactions. Indeed, depletion of acetyl CoA has been recently shown to be sufficient for autophagy induction and lifespan extension, although it is not known whether these effects are dependent on epigenetic changes (Eisenberg etਊl., 2014 Marino etਊl., 2014).

In humans, only nontoxic natural substances such as spermidine or resveratrol, which lead to deacetylation of chromatin, should be considered for clinical testing (Morselli etਊl., 2011). As a caveat, mechanistic understanding of this strategy is highly challenging as the drugs could have many off-target effects and even at the epigenetic level, the integrated response of multiple histone sites might be needed to mediate anti-aging effects. However, data from mice and humans indicate that spermidine has the potential to be safe for testing its epigenetic-dependent and independent effects on human healthspan. In one human study, a polyamine-rich traditional Japanese food (fermented soybeans) showed significant enhancement of polyamine concentration in the blood of the participants without obvious adverse effects (Soda etਊl., 2009).

Does Sirtuin protein family Sir2 work in low-calorie diet mostly? - Biology

Chapter 20 Utilizing Calorie Restriction to Evaluate the Role of Sirtuins in Healthspan and Lifespan of Mice Jessica Curtis and Rafael de Cabo Abstract Calorie restriction is the most powerful method currently known to delay aging-associated disease and extend lifespan. Use of this technique in combination with genetic models has led to identification of key metabolic regulators of lifespan. Limiting energy availability by restricting caloric intake leads to redistribution of energy expenditure and storage. The signaling required for these metabolic changes is mediated in part by the sirtuins at both the posttranslational and transcriptional levels, and consequently, sirtuins are recognized as instigating factors in the regulation of lifespan. This family of class III protein deacetylases is responsible for directing energy regulation based on NAD+ availability. However, there are many effectors of NAD+ availability, and hence sirtuin action, that should be considered when performing experiments using calorie restriction. The methods outlined in this chapter are intended to provide a guide to help the aging community to use and interpret experimental calorie restriction properly. The importance of healthspan and the use of repeated measures to assess metabolic health during lifespan experiments are strongly emphasized. Key words Sirtuins, Calorie restriction, Lifespan, Healthspan

Introduction In most animal models, limiting available energy by reducing caloric intake improves many metabolic health parameters, delaying onset of cancer, diabetes, cataracts, hypertension, and dyslipidemia [1–3]. It also enhances neuroprotection against age-related decline of cognitive function, stroke, and neurodegenerative diseases [4–6]. Consequently, calorie restriction (CR) serves as a strong positive effector on lifespan. As early as the 1930s, laboratory models of CR demonstrated extended lifespan in rodents exposed to a low calorie diet, predominantly due to reduced incidence of cancer [7]. Since then, efforts of CR studies have focused on elucidating the mechanism by which CR enhances lifespan and developing genetic models or pharmaceutical CR mimetics. Several molecular candidates have been implicated as effectors of CR, such as insulin-like growth factor (IGF-1), mammalian

Matthew D. Hirschey (ed.), Sirtuins: Methods and Protocols, Methods in Molecular Biology, vol. 1077, DOI 10.1007/978-1-62703-637-5_20, © Springer Science+Business Media, LLC 2013

Jessica Curtis and Rafael de Cabo

target of rapamycin (mTOR), and silent mating-type information regulator (SIRT1) [8]. The commonality between these molecules is their regulation of bioenergetics. SIRT1 belongs to the sirtuin family of class III protein deacetylases and contributes to the coordination of energy metabolism by directing cellular processes in accordance with the availability of the metabolic currency, NAD+. Low energy conditions like fasting or exercise increase the NAD+/ NADH ratio leading to sirtuin activation and deacetylation of protein targets such as AMPK, PGC-1α, p53, FOXO, and NFκB [9]. Sirtuin-activated signaling promotes energy redistribution in the form of lipolysis in the adipocyte, gluconeogenesis in the liver, and insulin secretion from the pancreas [10–12]. These efforts to maintain bioenergetic homeostasis inherently preserve organismal survival, illuminating an intimate relationship between energy sensing and longevity. The essential role of SIRT1 in mediating the bioenergetic effects of CR is still controversial, which may be due to subtle experimental variations. In lower organisms, such as yeast and worms, the SIRT1 orthologue Sir2 is required for CR to extend lifespan [13, 14]. CR induces SIRT1 expression in a variety of rodent tissues as well as in human cells treated with serum from CR rats [15]. SIRT1 null mice do not have extended lifespan when maintained on a CR diet for 10 months [16]. However, they also present with developmental defects and only survive on an outbred genetic background [17, 18]. These developmental issues may confound the interpretation of lifespan extension. The predominant evolutionary theory for CR-mediated lifespan extension is that survival in times of nutritional stress depended on effective shifts in energetic usage [8]. Therefore, it is easy to envisage that SIRT1 null mice may possess the stress pathways relevant to confer CR benefits but developmental defects inundate these overlapping signals, preventing the manifestation of benefits. Conditional SIRT1 knockout models will provide better evidence for the effects of SIRT1 on CR-mediated longevity by bypassing problems associated with loss of SIRT1 expression during embryonic and adolescent development [17]. Gain-of-function data on the role of SIRT1 in healthspan is notably less deniable. Whole-body SIRT1 transgenic mice exhibit the beneficial qualities invoked by the CR regimen such as lean body composition, reduced blood insulin and glucose levels, reduced levels of DNA damage, and decreased incidence of cancer but not extended lifespan [19, 20]. Furthermore, with the added stressor of a high-fat diet, SIRT1 overexpression protects against diet-induced metabolic damage [21], and activation of SIRT1 by

Utilizing Calorie Restriction to Evaluate the Role of Sirtuins in Healthspan…

pharmacological treatments, such as SRT1720, leads to extended healthspan and survival under high-fat diet conditions [22]. The survival effects of CR are not universal. Recently our lab has demonstrated in a longitudinal study that CR in rhesus monkeys does not extend survival [23], contrasting results of a previously published monkey study showing that CR did extend lifespan [24]. This outcome is speculated to arise from differences in diet composition, genetic background, feeding patterns, and statistical analysis. These publications highlight the importance of complete methodological reporting in the literature. There are many factors to consider when interpreting results of a CR experiment including circadian rhythm, diet composition, animal enrichment, and background strain. Many biomarkers of aging have been proposed in the field of aging, but lifespan analysis remains the gold standard. This is an easily measurable parameter and typically is demonstrated by a Kaplan–Meier curve. However, quality of life is as important as length of life, and in rodent models this is more difficult to characterize. Definitions of healthspan vary depending on the focus of each given research project, but generally, healthspan is considered to be the time that passes prior to the onset of age-related diseases. In humans, this can be monitored by parameters such as body-mass index, glucose tolerance, serum triglycerides, blood pressure, pulse wave velocity, bone density, waist:hip ratios, or even visual accommodation and a variety of mild physical stress tests, i.e., progressively inclined treadmill performance. These measures are tracked through the life of a patient and indicate when health issues arrive. Regardless of the parameters selected, in order to properly define healthspan in any particular model, it is crucial to have repeated measures of health parameters to identify the onset of impairments. Healthspan cannot be assessed by endpoint analysis. A longitudinal approach detailing metabolic alterations must be used to monitor metabolic health throughout the lifetime of an organism. No single panel of parameters characterizing healthspan has been defined, and measurements of healthspan are so inconsistent in the literature that meta-analyses and inter-study comparisons are difficult or impossible. Standardization of these measures is needed to ensure congruency between reports concerning healthspan. Our lab has developed a longitudinal approach for lifespan studies that integrates repeated measures to assess healthspan in addition to lifespan (Fig. 1). The methods outlined in this paper can be used to provide a more complete analysis of healthy aging, and specifically, how to properly use the CR paradigm in mice.

Jessica Curtis and Rafael de Cabo

Fig. 1 Testing interventions for longitudinal assessment of healthspan and lifespan. In order to determine the effect of the intervention on healthspan and lifespan, data should be collected at regular intervals throughout the study

1. Scale to weigh food and animals. 2. Mouse housing cages (see Note 1). 3. Purified diet (see Note 2).

Animal care and handling should be performed strictly according to approved institutional animal care protocols. Twelve-month-old C57Bl/6 mice are often the type of animals used in CR studies, but various ages, strain backgrounds, and genetic modification have also been used in published studies (see Notes 3 and 4).

3.1 Monitoring Food Consumption in Ad Libitum Animals

1. Place pre-weighed food in the hopper (see Notes 5 and 6). 2. After 7 days, weigh the food remaining in the hopper and calculate the food consumed per animal per day. 3. Monitor food consumption in AL animals for duration of the study or until the body weights of the CR cohort plateaus for 2–3 weeks. As the AL animals become moribund or die, it is customary to fix the CR intake at the final, accurately measured

Utilizing Calorie Restriction to Evaluate the Role of Sirtuins in Healthspan…

level (Basing the CR intake on that of a small group of moribund AL controls is inappropriate). 3.2 Calorie Restriction

1. Monitor food consumption and body weights weekly in control and experimental mice for at least 2 weeks before starting food restriction (see Note 7). 2. Once food consumption has stabilized, calculate food weights for CR cohort from the weekly food consumption of AL controls. Use a step-down approach for restriction so that animals can adjust to reduced calorie availability. If performing 40 % CR, start with 20 % CR for week one, 30 % CR during week two, and proceed to 40 % CR at week three. If animal health declines, extend step-down time or consider a lower percentage of caloric restriction. 3. Feed CR animals on the floor at the same time each day (see Note 8). Our mice are typically fed at the beginning of the light cycle. 4. Body weight of CR animals should plateau after approximately 10–15 weeks on the diet. Once this plateau is reached, continue feeding the CR animals a constant food allotment for the duration of the study.

3.3 Healthspan Analysis (Fig. 1)

1. Food intake and body weights should be collected biweekly for the duration of the study. The influence of a full stomach should be taken into account, since CR animals tend to gorge when presented with food. 2. Core body temperature can be monitored biweekly for the duration of the study with the use of implantable transponders (such as IPTT-300, BioMedic Data Systems). 3. Oral glucose and insulin tolerance tests should be performed every 6–12 months for the duration of the study [25]. 4. Body composition by NMR or dual-energy X-ray absorptiometry and indirect calorimetry (metabolic cages) should be performed every 6–12 months for the duration of the study [26]. 5. Physical performance assays (homecage activity, treadmill, strength tests) and behavioral testing (rotarod, open field, morris water maze) should be performed every 6–12 months for the duration of the study. 6. In a small subset of animals separate from the lifespan study, perform relevant immunological stress tests, such as the injection of LPS, tumor cells, or cold stress.

3.4 Lifespan Analysis (Fig. 2)

1. Viability should be monitored daily and as the animals advance in age the mice should be examined regularly (1–2 weeks). If an animal appears moribund, it should be euthanized per institutional protocols. Symptoms of moribund mice include lack

Jessica Curtis and Rafael de Cabo

Fig. 2 Interpreting intervention effects with Kaplan–Meier survival analysis. Interventions can have several effects on the shape of the survival curves, plotted as percent survival versus age. Average lifespan (solid black line) and extended lifespan (dashed black line) are depicted, where the shape is similar but the maximal lifespan is increased. Extending the healthspan of a cohort may be independent of lifespan extension and manifest as less early deaths (solid gray line). Animal cohorts with ideal healthspan would experience very few deaths before the maximum survival period (dashed gray line)

of response to touch stimuli, hunched spine, sudden weight loss, matted fur, slowed breathing, and lack of appetite. 2. As animals expire throughout the study, complete a pathological necropsy as soon as possible, recording observations of the external appearance of the animals including hair color and evidence of kyphosis (hunched spine). Preserve the heart, lungs, liver, spleen, kidneys, seminal vesicles, and bladder in 4 % formalin. Take note of enlarged, discolored, or tumorladen tissues. 3. Maximal lifespan is calculated as the mean lifespan of the top 10 % of survivors. 4. Median lifespan is calculated as the age at which 50 % of animals in a cohort survive. 5. Lifespan of control animals should be consistent between studies.

Notes 1. Population density should be considered when designing a CR study. The mouse is a social creature and prefers grouphousing conditions, but this can lead to increased variation in

Utilizing Calorie Restriction to Evaluate the Role of Sirtuins in Healthspan…

body weights and food consumption due to social relationships. It is certainly easier and cheaper for the researcher to use group housing, but it is important to be aware of the effects of housing density. Singly housed, C57Bl/6J male mice eat

15 % more than group housed animals on average due to lack of social conflicts (Dawn Boyer and Rafael de Cabo, unpublished). These results are after 24 weeks on diet, and no significant changes in body weight have been observed yet. The number of mice per cage will depend on the size of the cage, but delivery of food to animals should consider the number of animals in a cage. For example, if there are four animals in the cage, at least four pellets of food should be delivered to help alleviate social behavioral effects of food consumption. Our lab uses Thoren caging model #15 for group housing (four male adult mice preferred) and model #5 for single housing. 2. Using a purified, chemically defined diet is essential for experimental standardization and reproducibility. These diets come as hard packed pellets and nutrient formulation is independent of material sourcing or batch, which can vary in naturally sourced “chow” diets. The American Institute of Nutrition (AIN) provides many open source formulations, such as the AIN-93 series, that are suitable for CR studies. These diets may not provide trace nutrients found in naturally sourced diets. Addition of pharmacological compounds to the food may decrease palatability and inadvertently induce voluntary CR. When testing fooddelivered compounds, it is important to include a control-diet cohort to ensure that food consumption has not changed due to food taste. It is also important to realize that differing body composition between the control and CR animals influences micronutrient utilization and storage, particularly if a study will consider such things as lipid peroxidation (which is influenced by the ratio of adipose tissue to fat-soluble antioxidant intake). 3. The power calculation estimates the sample size needed to obtain significance dependent upon the observed change between variables and standard deviation. For example, 100 animals will provide approximately 100 % statistical power to observe a 10 % change in average lifespan with an assumed 10 % standard deviation, but using 50 animals drops the statistical power to approximately 80 %. Additional mice should be set up in parallel to the lifespan study, typically 6–12 animals, in order to collect tissues at various time points throughout the experiment or perform testing that may interfere with the lifespan study, such as immune challenges or cancer resistance. 4. Longevity effects are proportional to the duration and proportion of CR regardless of when CR was initiated. In a compilation of 24 published survival studies, the increase in survival appears to be inversely proportional to the decrease in calories [27].

Jessica Curtis and Rafael de Cabo

5. The mode of food delivery has an impact on the amount of food consumed. Delivery of food in the wire cage hopper reduces food consumption by more than 10 % (Dawn Boyer and Rafael de Cabo, unpublished). Our CR mice are typically floor fed. 6. The amount of food spilled into bedding does not differ between the CR and AL groups however, the amount of food spilled does increase with age leading to an overestimation of food consumption [28]. Food spillage is also dependent upon the composition of the food and possibly the genetic or strain background of the animal. 7. Genetically modified animals may present with differences in food consumption, possibly due to changes in body weight. It is important to have an AL control for each experimental variable. 8. Gene expression and metabolic regulation are coordinately regulated by circadian rhythm. AL mice tend to eat during periods of darkness, while CR mice will consume their food at the time it is provided. Furthermore, in CR cohorts, the changes between post-prandial and post-absorptive states become exaggerated. Circadian rhythms usually differ between AL and CR groups, and accidental comparisons between fasted, active, and hungry cohorts and somnolent, inactive, just-fed cohorts should be avoided. It is important to be consistent with feeding time in your study and to report the time of sample collection relative to each cohort’s diurnal cycle.

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