Those of you who have put together jigsaw puzzles know that every once in a while a piece is found that links together several seemingly unrelated chunks of the puzzle. The sirtuin SIRT3 is doing that for several chunks of the aging/longevity puzzle, showing a key way in which the Oxidative Damageand the Mitochondrial Damagetheories of aging fit together and how these fit with the known life-extending properties of calorie restriction, with the role of exercise, response to stress and PGC – 1alpha, cell metabolism, several age-related diseases including obesity, diabetes and Alzheimer’s Disease, the FOXO gene family, and actions of P53, and with the actions of the dietary polyphenol resveratrol. Much of the relevant research is quite recent.
(From the introduction to Mitochondrial SIRT3 and heart disease) “The desire to live longer and probably forever has long fascinated mankind. Concoctions to prevent ageing and maintain youth have been described in medical books of ancient civilizations, including Charaka Samhita, the most ancient textbook of Ayurveda (an Indian system of traditional medicine), which is believed to have been written centuries before the birth of Christ. It seems our forefathers found a way to live longer and healthy by undergoing calorie restriction, a diet regimen that is considered to be impractical for modern society where food is surplus and time is scarce. Even though vaccination, antibiotics, better child care, and early disease-detection techniques in combination with modern drugs have helped us to increase our average lifespan, the quest to increase maximal lifespan still remains elusive. The major advances in ageing research that we have witnessed in the past two decades are the rediscovery of benefits of calorie restriction, and the delineation of the molecular mechanism involved in its protective effects. Many studies have proposed that the beneficial effect of calorie restriction is mediated through a set of genes collectively called sirtuins (SIRT1–7).1”
Mitochondrial sirtuins – focus on SIRT3
In humans, there are at least seven sirtuins (SIRT1–7), proteins with diverse actions including the regulation of metabolism and chromatin structure, DNA repair and preservation of genomic integrity. I have discussed actions of SIRT1 and SIRT6 in several previous blog entries (ref)(ref)(ref)(ref). Lifespan extension has been linked to actions of sirtuins in various publications(ref).
SIRT3 is a mitochondrial protein that serves to deacetylate acetyllysine-modified proteins in mitochondria
From the 2010 publication Mitochondrial sirtuins “Three sirtuins, SIRT3, 4 and 5, are located within the mitochondrial matrix. SIRT3 and SIRT5 are NAD(+)-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins and yield 2′-O-acetyl-ADP-ribose and nicotinamide. SIRT4 can transfer the ADP-ribose group from NAD(+) onto acceptor proteins. Recent findings reveal that a large fraction of mitochondrial proteins are acetylated and that mitochondrial protein acetylation is modulated by nutritional status. This and the identification of targets for SIRT3, 4 and 5 support the model that mitochondrial sirtuins are metabolic sensors that modulate the activity of metabolic enzymes via protein deacetylation or mono-ADP-ribosylation.”
The 2007 publication Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation reports “Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.”
Expression of SIRT3 is controlled by diet and exercise
The 2009 publication Diet and exercise signals regulate SIRT3 and activate AMPK and PGC-1alpha in skeletal muscle reports “SIRT3 is a member of the sirtuin family of NAD(+)-dependent deacetylases, which is localized to the mitochondria and is enriched in kidney, brown adipose tissue, heart, and other metabolically active tissues. We report here that SIRT3 responds dynamically to both exercise and nutritional signals in skeletal muscle to coordinate downstream molecular responses. We show that exercise training increases SIRT3 expression as well as associated CREB phosphorylation and PGC-1alpha up-regulation. Furthermore, we show that SIRT3 is more highly expressed in slow oxidative type I soleus muscle compared to fast type II extensor digitorum longus or gastrocnemius muscles. Additionally, we find that SIRT3 protein levels in skeletal muscle are sensitive to diet, for SIRT3 expression increases by fasting and caloric restriction, yet it is decreased by high-fat diet. Interestingly, the caloric restriction regimen also leads to phospho-activation of AMPK in muscle. Conversely in SIRT3 knockout mice, we find that the phosphorylation of both AMPK and CREB and the expression of PGC-1alpha are down regulated, suggesting that these key cellular factors may be important components of SIRT3-mediated biological signals in vivo.”
Cellular stress causes SIRT3 to translocate from the nucleus to the mitochondria and to be highly expressed in brown adipose tissue
The 2007 publication SirT3 is a nuclear NAD+-dependent histone deacetylase that translocates to the mitochondria upon cellular stress reports “SirT3 levels have been shown to correlate with extended life span, to localize to the mitochondria, and to be highly expressed in brown adipose tissue. — In humans, SirT3 exists in two forms, a full-length protein of approximately 44 kDa and a processed polypeptide lacking 142 amino acids at its N terminus. We found that SirT3 not only localizes to the mitochondria, but also to the nucleus under normal cell growth conditions. Both the full-length and processed forms of SirT3 target H4-K16 for deacetylation in vitro and can deacetylate H4-K16 in vivo when recruited to a gene. Using a highly specific antibody against the N terminus of SirT3, we found that SirT3 is transported from the nucleus to the mitochondria upon cellular stress. This includes DNA damage induced by Etoposide and UV-irradiation, as well as overexpression of SirT3 itself.”
There are two isoforms of SIRT3 (in mice at least) with somewhat different properties
As time progresses more and more of the detailed structure and workings of SIRT3 are being discovered. The 2010 publication Characterization of the murine SIRT3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short SIRT3 isoforms relates “SIRT3 is identified as the major mitochondrial deacetylase. Two distinct isoforms of the murine SIRT3 have been identified with the short isoform having no recognizable mitochondrial localization sequence (MLS) and the long isoform having a putative MLS. A recent study questions the mitochondrial deacetylase activity of this short isoform. In contrast, the long isoform has been shown to be predominantly mitochondrial with robust deacetylase activity. In this study, we investigate whether the amino-terminus of the long SIRT3 isoform is a legitimate MLS and evaluate in-situ mitochondrial deacetylase activity of both isoforms. We confirm the presence of long and short isoforms in murine liver and kidney. — Despite lower mitochondrial expression of the short isoform, the capacity to deacetylate mitochondrial proteins and to restore mitochondrial respiration is equally robust following transient transfection of either isoform into SIRT3 knockout embryonic fibroblasts. How these alternative transcripts are regulated and whether they modulate distinct targets is unknown. Furthermore, in contrast to exclusive mitochondrial enrichment of endogenous SIRT3, overexpression of both isoforms shows nuclear localization. This overexpression effect, may partially account for previously observed divergent phenotypes attributed to SIRT3.”
PGC1-alpha is an upstream activator of SIRT3. Further, SIRT3 suppresses mitochondrial ROS and promotes mitochondrial biogenesis
As discussed in the blog entry PGC-1alpha and exercise, the protein PGC1-alpha (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) appears to be the mediator of the health benefits produced by exercise, and plays an important role in the metabolism of both white and brown fat. PGC1-alpha also plays a role in the the regulation of mitochondrial biogenesis, and is a major factor that regulates muscle fiber type determination. This protein also appears to be implicated in the regulation of cellular cholesterol homoeostasis, control of blood pressure, and the development of obesity.
The 2010 publication Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS and mitochondrial biogenesisreports “(PGC-1alpha) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1alpha induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood. — RESULTS: Here we show that PGC-1alpha strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. — Furthermore, Sirt3 was essential for PGC-1alpha-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1alpha in C(2)C(12) myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1alpha on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2)C(12) myotubes. — CONCLUSION: Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation.”
Calorie restriction results in increased expression of SIRT3 which produces a stronger mitochondrial defense against free radicals resulting in mammals living longer
A November 2010 publication Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-Related Hearing Loss under Caloric Restriction links SIRT3 to the longevity benefits of calorie restriction. “Here, we report that CR reduces oxidative DNA damage in multiple tissues and prevents AHL in wild-type mice but fails to modify these phenotypes in mice lacking the mitochondrial deacetylase Sirt3, a member of the sirtuin family. In response to CR, Sirt3 directly deacetylates and activates mitochondrial isocitrate dehydrogenase 2 (Idh2), leading to increased NADPH levels and an increased ratio of reduced-to-oxidized glutathione in mitochondria. In cultured cells, overexpression of Sirt3 and/or Idh2 increases NADPH levels and protects from oxidative stress-induced cell death. Therefore, our findings identify Sirt3 as an essential player in enhancing the mitochondrial glutathione antioxidant defense system during CR and suggest that Sirt3-dependent mitochondrial adaptations may be a central mechanism of aging retardation in mammals.”
Thus, actions of SIRT3 link up the Oxidative Damageand the Mitochondrial Damagetheories of aging. The research strongly suggests that minimizing mitochondrial oxidative damage can extend life spans. Further, three practical sirtuin-related ways of extending life spans appear to be 1. increasing expression of SIRT3 via exercise, 2. Promotion of expression of SIRT1 via resveratrol and 3. calorie restriction which appears to affect both SIRT1 and SIRT3.
According to the November 2010 Science Daily report on this research Scientists Ferret out a Key Pathway for Aging, “It has been well documented in species ranging from spiders to monkeys that a diet with consistently fewer calories can dramatically slow the process of aging and improve health in old age. But how a reduced diet acts at the most basic level to influence metabolism and physiology to blunt the age-related decline of tissues and cells has remained, for the most part, a mystery. — Now, writing in the Nov. 18 online issue of the journal Cell, a team of scientists from the University of Wisconsin-Madison and their colleagues describe a molecular pathway that is a key determinant of the aging process. The finding not only helps explain the cascade of events that contributes to aging, but also provides a rational basis for devising interventions, drugs that may retard aging and contribute to better health in old age. — “We’re getting closer and closer to a good understanding of how caloric restriction works,” says Tomas A. Prolla, a UW-Madison professor of genetics and a senior author of the new Cell study. “This study is the first direct proof for a mechanism underlying the anti-aging effects we observe under caloric restriction.” — The Wisconsin study focuses on an enzyme known as Sirt3, one of a family of enzymes known as sirtuins, which have been implicated in previous studies in the aging process, gene transcription, programmed cell death and stress resistance under reduced calorie conditions. In mammals, including humans, there are seven sirtuins that seem to have wide-ranging influence on cell fate and physiology. — Sirt3 has been less studied than other members of the sirtuin family, but the new study provides “the first clear evidence that sirtuins have anti-aging effects in mammals,” according to John M. Denu of UW-Madison’s Wisconsin Institute for Discovery and a senior author of the report. — The Sirt3 enzyme, Denu explains, acts on mitochondria, structures inside cells that produce energy and that are the sources of highly reactive forms of oxygen known as free radicals, which damage cells and promote the effects of aging. Under reduced-calorie conditions, levels of Sirt3 amp up, altering metabolism and resulting in fewer free radicals produced by mitochondria. — “This is the strongest and most direct link that caloric restriction acts through mitochondria,” says Prolla, who has studied the effects of reduced calorie diets on aging and health for more than a decade. “Sirt3 is playing a surprisingly important role in reprogramming mitochondria to deal with an altered metabolic state under caloric restriction.”
SIRT3 regulates P53-induced cell senescence
The 2010 publication p53-induced growth arrest is regulated by the mitochondrial SirT3 deacetylasereports “A hallmark of p53 function is to regulate a transcriptional program in response to extracellular and intracellular stress that directs cell cycle arrest, apoptosis, and cellular senescence. Independent of the role of p53 in the nucleus, some of the anti-proliferative functions of p53 reside within the mitochondria . p53 can arrest cell growth in response to mitochondrial p53 in an EJ bladder carcinoma cell environment that is naÃ¯ve of p53 function until induced to express p53 . TP53 can independently partition with endogenous nuclear and mitochondrial proteins consistent with the ability of p53 to enact senescence.In order to address the role of p53 in navigating cellular senescence through the mitochondria, we identified SirT3 to rescue EJ/p53 cells from induced p53-mediated growth arrest. Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis. Our evidence suggests that SirT3 partially abrogates p53 activity to enact growth arrest and senescence. Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence. These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.”
SIRT3 protects in-vitro fertilized embryos against P53-mediated developmental arrest induced by oxidative stress
According to the 2010 publication Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest, “When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in vitro conditions and that the effect was long lasting. Further experiments revealed that maternally derived Sirt3 was critical. Sirt3 inactivation increased mitochondrial ROS production, leading to p53 upregulation and changes in downstream gene expression. The inactivation of p53 improved the developmental outcome of Sirt3-knockdown embryos, indicating that the ROS-p53 pathway was responsible for the developmental defects. These results indicate that Sirt3 plays a protective role in preimplantation embryos against stress conditions during in vitro fertilization and culture.”
SIRT3 affects gene expression in two longevity-related gene families: the sirtuins and FOXO
The 2008 publication SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a dependent gene expressionrelates “Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.”
SIRT3 blocks the cardiac hypertrophic response in mice via a FOXO-related pathway
The 2009 paper Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice states “Sirtuin 3 (SIRT3) is a member of the sirtuin family of proteins that promote longevity in many organisms. Increased expression of SIRT3 has been linked to an extended life span in humans. Here, we have shown that Sirt3 protects the mouse heart by blocking the cardiac hypertrophic response. Although Sirt3-deficient mice appeared to have normal activity, they showed signs of cardiac hypertrophy and interstitial fibrosis at 8 weeks of age. Application of hypertrophic stimuli to these mice produced a severe cardiac hypertrophic response, whereas Sirt3-expressing Tg mice were protected from similar stimuli. In primary cultures of cardiomyocytes, Sirt3 blocked cardiac hypertrophy by activating the forkhead box O3a-dependent (Foxo3a-dependent), antioxidant-encoding genes manganese superoxide dismutase (MnSOD) and catalase (Cat), thereby decreasing cellular levels of ROS. Reduced ROS levels suppressed Ras activation and downstream signaling through the MAPK/ERK and PI3K/Akt pathways. This resulted in repressed activity of transcription factors, specifically GATA4 and NFAT, and translation factors, specifically eukaryotic initiation factor 4E (elf4E) and S6 ribosomal protein (S6P), which are involved in the development of cardiac hypertrophy. These results demonstrate that SIRT3 is an endogenous negative regulator of cardiac hypertrophy, which protects hearts by suppressing cellular levels of ROS.”
SIRT3 is a tumor suppressor
The 2010 comment in the Journal Cancer Cell A tumor suppressor SIRTainty outlines the story: “Sirtuin deacetylases are linked to longevity, aging, and stress responses. In this issue of Cancer Cell, Kim et al. show that SIRT3 functions as a tumor suppressor by enhancing the expression of mitochondrial MnSOD. Loss of SIRT3 leads to increased mitochondrial ROS, which then enhances cellular transformation and tumor growth.” The Kim et al. publication is SIRT3 is a mitochondria-localized tumor suppressor required for maintenance of mitochondrial integrity and metabolism during stress. “This work demonstrates that SIRT3(-/-) mouse embryonic fibroblasts (MEFs) exhibit abnormal mitochondrial physiology as well as increases in stress-induced superoxide levels and genomic instability. Expression of a single oncogene (Myc or Ras) in SIRT3(-/-) MEFs results in in vitro transformation and altered intracellular metabolism. Superoxide dismutase prevents transformation by a single oncogene in SIRT3(-/-) MEFs and reverses the tumor-permissive phenotype as well as stress-induced genomic instability. In addition, SIRT3(-/-) mice develop ER/PR-positive mammary tumors. Finally, human breast and other human cancer specimens exhibit reduced SIRT3 levels. These results identify SIRT3 as a genomically expressed, mitochondria-localized tumor suppressor.”
I comment that this work is coincident with a rising focus on mitochondrial ROS damage as a possible and even probable cause for cancer as outlined in the 2010 publicationThe causes of cancer revisited: “mitochondrial malignancy” and ROS-induced oncogenic transformation – why mitochondria are targets for cancer therapy. “— more recent evidence indicates the importance of two additional key factors imposed on proliferating cells that are involved in transformation to malignancy and these are hypoxia and/or stressful conditions of nutrient deprivation (e.g. lack of glucose). These two additional triggers can initiate and promote the process of malignant transformation when a low percentage of cells overcome and escape cellular senescence. It is becoming apparent that hypoxia causes the progressive elevation in mitochondrial ROS production (chronic ROS) which over time leads to stabilization of cells via increased HIF-2alpha expression, enabling cells to survive with sustained levels of elevated ROS. –. Recent evidence also indicates that the resulting mutated cancer-causing proteins feedback to amplify the process by directly affecting mitochondrial function in combinatorial ways that intersect to play a major role in promoting a vicious spiral of malignant cell transformation. Consequently, many malignant processes involve periods of increased mitochondrial ROS production when a few cells survive the more common process of oxidative damage induced cell senescence and death. The few cells escaping elimination emerge with oncogenic mutations and survive to become immortalized tumors. — ”
SIRT3 plays an important role in cardioprotection
The 2008 publication SIRT3 Is a Stress-Responsive Deacetylase in Cardiomyocytes That Protects Cells from Stress-Mediated Cell Death by Deacetylation of Ku70 relates “We show that, like human SIRT3, mouse SIRT3 is expressed in two forms, a 44-kDa long form and a 28-kDa short form. Whereas the long form is localized in the mitochondria, nucleus, and cytoplasm, the short form is localized exclusively in the mitochondria of cardiomyocytes. During stress, SIRT3 levels are increased not only in mitochondria but also in the nuclei of cardiomyocytes. We also identified Ku70 as a new target of SIRT3. SIRT3 physically binds to Ku70 and deacetylates it, and this promotes interaction of Ku70 with the proapoptotic protein Bax. Thus, under stress conditions, increased expression of SIRT3 protects cardiomyocytes, in part by hindering the translocation of Bax to mitochondria.”
The 2010 publication Mitochondrial SIRT3 and heart disease reports “Although the role of SIRT3 in cell biology is only beginning to be understood, initial studies have shown that SIRT3 plays a major role in free fatty acid oxidation and maintenance of cellular ATP levels. In the heart SIRT3 has been found to block development of cardiac hypertrophy and protect cardiomyocytes from oxidative stress-mediated cell death. Similarly, SIRT3 has been reported to have tumour-suppressive characteristics. In this article, we review the known effects of SIRT3 in different tissues and relate them to the protection of cardiomyocytes under stress.”
thanks for sharing your research. will be in touch while reading your information.
Hello Vincent, I recently found this you tube video that I think you will find interesting. This Russian researcher claims to have found the Fountain of Youth. I am skeptical of such claims, but his research does tie in with the oxidative damage theory of aging. Also, have you had time to look into PQQ? Thanks Vincent.
Thanks for the clue. I will check out the video and follow-through. Out-of-the-box I share your skepticism, but I am always open to pleasant surprises.
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