17 Jun 2004

Sir Lancelot, for all his prowess protecting King Arthur’s court, couldn’t do one thing: prevent the death toll extracted by living high at the round table. Sir2 could have shown our legendary hero how to fight that battle gallantly. In today’s Sciencexpress, David Sinclair and colleagues at Harvard Medical School report that the mammalian protein SIRT1 promotes the survival of mammalian cells and suggest it may do so by preventing apoptosis. Like Sir2, SIRT1 is induced by caloric restriction (CR) and the actions of this protein open a door toward a better understanding of why CR delays the onset of numerous age-associated diseases, including Alzheimer’s disease, which has been linked to high caloric intake (see ARF related news story).

In yeast, caloric restriction leads to induction of the Sir2 histone deacetylase and subsequent gene silencing (see ARF related news story). To test if caloric restriction induces the Sir2 homolog SIRT1 in mammals, first author Haim Cohen and colleagues examined tissues extracted from year-old rats that had been fed either ad libitum, or a calorie-restricted diet (these animals ate about 40 percent less than their littermates). Cohen found that CR led to a greater abundance of SIRT1 in a variety of tissues including brain, liver, kidney and fat. In humans, CR may elicit similar changes because when Cohen grew human embryonic kidney cells (293T cells) in serum from CR rats, SIRT1 expression doubled.

So what does SIRT1 do in mammals? Work at Leonard Guarente’s lab at MIT has shown that SIRT1 can silence apoptotic forkhead transcription factors, such as Foxo3a (see Motta et al., 2004). But Sinclair and colleagues have found that SIRT1 can prevent apoptosis through an alternative pathway involving Bax, a protein which, when translocated to the outer mitochondrial membrane, triggers the release of cytochrome C, an apoptotic trigger. When Cohen examined 293T cells grown in serum from calorie-restricted rats, he found that they were less susceptible to Bax-mediated apoptosis.

Bax is normally sequestered in the cytosol by the protein Ku70. Acetylation of Ku70 releases Bax and pushes cells down the slippery apoptotic slope (see Cohen et al., 2004). Sinclair and colleagues wondered if the deacetylase activity of SIRT1 was the glue that holds Bax and Ku70 together. To test this, Cohen measured, in a test tube, deacetylation of Ku70 by SIRT1. Indeed, the authors found the SIRT1 could effectively remove acetyl groups from two Ku70 lysine residues that are essential for binding Bax. Similar results were obtained when Cohen measured acetylation of Ku70 in cells expressing normal or dominant-negative mutants of SIRT1.

All told, the experiments suggest that caloric restriction induces expression of the deacetylase, which then prevents acetylation of Ku70 and so retains Bax in the cytosol, where it does no damage. What component of rat CR-conditioned serum induces expression of SIRT1? While the scientists puzzle out this question, what’s a bon vivant to do? Very few people manage to stay on a caloric restriction for long, Mark Mattson being a famous exception from within the AD field. Maybe we can take one well-chosen leaf out of King Arthur’s book and live it up just a little. Recent findings from Sinclair’s group have shown the resveratrol, a polyphenol found in red wine, activates SIRT1. In yeast the polyphenol can increase life span by 70 percent (see Howitz et al., 2004). Other groups have sung resveratrol's praises as an antioxidant. A tankard, anyone?—Tom Fagan

Q&A with David Sinclair

Q: At first blush, Ku70 brings to mind DNA repair. Could SIRT1's deacetylation of Ku70 exert its life-prolonging effect by helping the cell repair DNA breaks acquired with age?
A: It is very possible that Ku70 helps extend lifespan by controlling DNA repair and cell death. This would make a lot of sense. We are testing this possibility now.

Q: Korean scientists last year reported that caloric restriction increased Ku activity in some aging tissues but not others (Um et al., 2003), though they unfortunately did not report on brain. Did you see such differences?
A: I believe they tested DNA repair capacity, which we have not yet tested. We also don't know the effect of CR on Ku70's ability to prevent cell death in vivo yet. It's hard to test.

Q: Did you see any induction of chaperones with CR?
A: See deCabo et al., 2003. They showed that CR upregulates heat shock protein 70, a chaperone. This is also induced by CR serum, as we show in our paper.

Q: Caloric restriction by 40 percent makes for pretty austere living. For those whose flesh is too weak for that, does cutting 10 or 20 percent help, also?
A: It depends on where you start. If you are eating a lean diet already, then a 10% drop might have some benefit, but not as great as 30-40 percent. Basically, for this to work, your body has to be in a state of hunger for most of the time.

Q: Does this depend on body weight? How about people who eat a lot but stay thin?
A: This doesn't work. Just being thin is not severe enough to turn on the body's defenses.

Q: Do you have a specific research interest in neurodegeneration?
A: Not yet, but we will soon be looking at whether the SIRT1 enzyme plays a protective role in neurons and whether the SIRT1-activating molecules (STACs) that we discovered last year can slow neurodegeneration in mice.

Comments

1. • Andrea LeBlanc
     McGill University
   • Posted: 17 Jun 2004

   The laboratory of David Sinclair shows an interesting feature of caloric restriction in this paper. Caloric restriction increases the levels of SIRT1, the mammalian counterpart of yeast Sir2, a nicotinamide adenosine dinucleotide-dependent histone deacetylase protein known to be involved in longevity mediated by caloric restriction. This effect can be mimicked in human embryonic kidney 293T cells by treating the cells with serum from calorie-restricted rats. This papers shows that SIRT1 deacetylates Ku70, allowing Ku70 to interact with the pro-apoptotic protein, Bax, and prevent Bax-mediated cell death. It is therefore suggested that calorie restriction extends lifespan through increased SIRT1 expression and promotion of survival of the organism’s irreplaceable cells.

   Coupled with a recent paper in Molecular Cell (Cohen et al., 2004), this group shows a very compelling mechanism for the regulation of Bax function through the acetylation and deacetylation of Ku70, a DNA repair protein, originally discovered by Shigemi Matsuyama, to be a Bax inhibitor (Sawada et al., 2003; Sawada et al., 2003). The suggestion that deacetylated Ku70 sequesters Bax from translocating to the mitochondria to initiate the release of apoptogenic factors such as cytochrome c, apoptosis inducing factor (AIF) or endonuclease G, remains to be shown. However, it is clear that deacetylated Ku70 eliminates Bax pro-apoptotic function.

   The suggestion that preventing Bax translocation to the mitochondria explains the extended lifespan of an animal by promoting survival of terminally differentiated cells is intriguing but remains to be proven. Alternatively, cytosolic Bax have a physiological function and retention in the cytosol with Ku70 could promote this function. Furthermore, acetylation and deacetylation of Ku70 could influence DNA repair and allow resistance to age-dependant DNA damage. If Bax and Ku70 are central to longevity, Bax or Ku70 null mice, which are viable, would also be expected to have a longer lifespan.

   Alternative possibilities also exist to explain SIRT1-mediated cell survival. SIRT1 deacetylates a number of other proteins including forkhead transcription factors and p53, two other proteins involved in the balance of cellular survival and cell death (Motta et al., 2004). In this paper, it is proposed that downregulation of forkhead factors and p53 increases the threshold for apoptosis and cellular senescence. Furthermore, these authors suggest that the regulation of forkhead-mediated transcription may alter diet-responsive metabolism thereby decreasing the level of oxidative stress and damage to cells. Therefore, it appears that SIRT1 can have multiple beneficial coordinated effects to control cellular survival and longevity.

   References:

   Cohen HY, Lavu S, Bitterman KJ, Hekking B, Imahiyerobo TA, Miller C, Frye R, Ploegh H, Kessler BM, Sinclair DA. Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax-mediated apoptosis. Mol Cell. 2004 Mar 12;13(5):627-38. PubMed.

   Sawada M, Hayes P, Matsuyama S. Cytoprotective membrane-permeable peptides designed from the Bax-binding domain of Ku70. Nat Cell Biol. 2003 Apr;5(4):352-7. PubMed. RETRACTED

   Sawada M, Sun W, Hayes P, Leskov K, Boothman DA, Matsuyama S. Ku70 suppresses the apoptotic translocation of Bax to mitochondria. Nat Cell Biol. 2003 Apr;5(4):320-9. PubMed. RETRACTED

   Motta MC, Divecha N, Lemieux M, Kamel C, Chen D, Gu W, Bultsma Y, McBurney M, Guarente L. Mammalian SIRT1 represses forkhead transcription factors. Cell. 2004 Feb 20;116(4):551-63. PubMed.

   View all comments by Andrea LeBlanc

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References

News Citations

1. Fat and Calories Mean Higher AD Risk 22 Nov 2002
2. Mean Life Expectancy 112? 3 Mar 2003

Paper Citations

1. Motta MC, Divecha N, Lemieux M, Kamel C, Chen D, Gu W, Bultsma Y, McBurney M, Guarente L. Mammalian SIRT1 represses forkhead transcription factors. Cell. 2004 Feb 20;116(4):551-63. PubMed.
2. Cohen HY, Lavu S, Bitterman KJ, Hekking B, Imahiyerobo TA, Miller C, Frye R, Ploegh H, Kessler BM, Sinclair DA. Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax-mediated apoptosis. Mol Cell. 2004 Mar 12;13(5):627-38. PubMed.
3. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, Zipkin RE, Chung P, Kisielewski A, Zhang LL, Scherer B, Sinclair DA. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature. 2003 Sep 11;425(6954):191-6. PubMed.
4. Um JH, Kim SJ, Kim DW, Ha MY, Jang JH, Chung BS, Kang CD, Kim SH. Tissue-specific changes of DNA repair protein Ku and mtHSP70 in aging rats and their retardation by caloric restriction. Mech Ageing Dev. 2003 Aug-Sep;124(8-9):967-75. PubMed.
5. de Cabo R, Fürer-Galbán S, Anson RM, Gilman C, Gorospe M, Lane MA. An in vitro model of caloric restriction. Exp Gerontol. 2003 Jun;38(6):631-9. PubMed.

Further Reading

Papers

1. Blander G, Guarente L. The Sir2 family of protein deacetylases. Annu Rev Biochem. 2004;73:417-35. PubMed.
2. Picard F, Kurtev M, Chung N, Topark-Ngarm A, Senawong T, Machado De Oliveira R, Leid M, McBurney MW, Guarente L. Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma. Nature. 2004 Jun 17;429(6993):771-6. PubMed.
3. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, Zipkin RE, Chung P, Kisielewski A, Zhang LL, Scherer B, Sinclair DA. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature. 2003 Sep 11;425(6954):191-6. PubMed.
4. Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H, Ross SE, Mostoslavsky R, Cohen HY, Hu LS, Cheng HL, Jedrychowski MP, Gygi SP, Sinclair DA, Alt FW, Greenberg ME. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science. 2004 Mar 26;303(5666):2011-5. PubMed.
5. Doria G, Barattini P, Scarpaci S, Puel A, Guidi L, Frasca D. Role of immune responsiveness and DNA repair capacity genes in ageing. Ageing Res Rev. 2004 Apr;3(2):143-51. PubMed.
6. Motta MC, Divecha N, Lemieux M, Kamel C, Chen D, Gu W, Bultsma Y, McBurney M, Guarente L. Mammalian SIRT1 represses forkhead transcription factors. Cell. 2004 Feb 20;116(4):551-63. PubMed.
7. Um JH, Kim SJ, Kim DW, Ha MY, Jang JH, Chung BS, Kang CD, Kim SH. Tissue-specific changes of DNA repair protein Ku and mtHSP70 in aging rats and their retardation by caloric restriction. Mech Ageing Dev. 2003 Aug-Sep;124(8-9):967-75. PubMed.