2 replies to this topic

[zoolander]

Here is the abstract

Mech Ageing Dev. 2006 Apr 5; [Epub ahead of print] 
   
    SIRT1 stimulation by polyphenols is affected by their stability and metabolism.

    de Boer VC, de Goffau MC, Arts IC, Hollman PC, Keijer J.

    RIKILT - Institute of Food Safety, Wageningen University and Research Centre, Wageningen, The Netherlands.

    Silent information regulator two ortholog 1 (SIRT1) is the human ortholog of the yeast sir2 protein; one of the most important regulators of lifespan extension by caloric restriction in several organisms. Dietary polyphenols, abundant in vegetables, fruits, cereals, wine and tea, were reported to stimulate the deacetylase activity of recombinant SIRT1 protein and could therefore be potential regulators of aging associated processes. However, inconsistent data between effects of polyphenols on the recombinant SIRT1 and on in vivo SIRT1, led us to investigate the influence of (1) stability of polyphenols under experimental conditions and (2) metabolism of polyphenols in human HT29 cells, on stimulation of SIRT1. With an improved SIRT1 deacetylation assay we found three new polyphenolic stimulators. Epigallocatechin galate (EGCg, 1.76-fold), epicatechin galate (ECg, 1.85-fold) and myricetin (3.19-fold) stimulated SIRT1 under stabilizing conditions, whereas without stabilization, these polyphenols strongly inhibited SIRT1, probably due to H(2)O(2) formation. Using metabolically active HT29 cells we were able to show that quercetin (a stimulator of recombinant SIRT1) could not stimulate intracellular SIRT1. The major quercetin metabolite in humans, quercetin 3-O-glucuronide, slightly inhibited the recombinant SIRT1 activity which explains the lack of stimulatory action of quercetin in HT29 cells. This study shows that the stimulation of SIRT1 is strongly affected by polyphenol stability and metabolism, therefore extrapolation of in vitro SIRT1 stimulation results to physiological effects should be done with caution.

    PMID: 16603228 [PubMed - as supplied by publisher]

Polyphenols are antioxidants and are readily oxidaized in aqueous media. For example, EGCg and gallic acid (200uM) produce up to ~650uM H2O2 when incubated for an hour in HEPES buffered DMEM medium. Therefore, these researchers used excess Vitamin C to stabilize H2O2 production. So when you read the term "stabilization" above you will know what they are refering to.

The problem with studies using cell culture is that the polyphenols do not undergo phase II glucoronidation in the liver. For example, Howitz et al. reports a 4.6 fold increase in the deacetylation activity of SIRT1 with quercetin, however in humans quercetin is rapidly metabolized via phase II glucoronidation to quercetin 3-O-glucoronide which did not stimulate the recombinant SIRT1 as reported in the above de BOER et al. study . Hence, they warn that [airquote] extrapolation of in vitro SIRT1 stimulation results to physiological effects should be done with caution. [/airquote]

So as usual, the news is good however we need to find ways to deliver the compounds effectively in biological systems. More human studies are needed.

Edited by zoolander, 24 May 2006 - 10:57 PM.

[opales]

Re:sirtuins

http://www.sciencema...t/309/5742/1861

Reports
HST2 Mediates SIR2-Independent Life-Span Extension by Calorie Restriction
Dudley W. Lamming,1* Magda Latorre-Esteves,1* Oliver Medvedik,1* Stacy N. Wong,2* Felicia A. Tsang,2 Chen Wang,2 Su-Ju Lin,2{dagger} David A. Sinclair1{dagger}

Calorie restriction (CR) extends the life span of numerous species, from yeast to rodents. Yeast Sir2 is a nicotinamide adenine dinucleotide (NAD+-dependent histone deacetylase that has been proposed to mediate the effects of CR. However, this hypothesis has been challenged by the observation that CR can extend yeast life span in the absence of Sir2. Here, we show that Sir2-independent life-span extension is mediated by Hst2, a Sir2 homolog that promotes the stability of repetitive ribosomal DNA, the same mechanism by which Sir2 extends life span. These findings demonstrate that the maintenance of DNA stability is critical for yeast life-span extension by CR and suggest that, in higher organisms, multiple members of the Sir2 family may regulate life span in response to diet.

1 Paul F. Glenn Laboratories for the Biological Mechanisms of Aging, Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
2 Center for Genetics and Development, and Section of Microbiology, University of California Davis, 351 Briggs Hall, Davis, CA 95616, USA.

Critical comment

http://www.sciencema...;312/5778/1312b

Comment on "HST2 Mediates SIR2-Independent Life-Span Extension by Calorie Restriction"
Matt Kaeberlein,1* Kristan K. Steffen,2 Di Hu,2 Nick Dang,2 Emily O. Kerr,2 Mitsuhiro Tsuchiya,2 Stanley Fields,3 Brian K. Kennedy2*

Calorie restriction (CR) increases life span in yeast independently of Sir2. Lamming et al. (Reports, 16 September 2005, p. 1861) recently proposed that Sir2-independent life-span extension by CR is mediated by the Sir2 paralogs Hst1 and Hst2. Contradictory to this, we find that CR greatly increases life span in cells lacking Sir2, Hst1, and Hst2, which suggests that CR is not mediated by Sir2, Hst2, or Hst1.

1 Department of Pathology, University of Washington, Seattle, WA 98195, USA.
2 Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
3 Departments of Genome Sciences and Medicine, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.

* To whom correspondence should be addressed. E-mail: kaeber@u.washington.edu (M.K.); bkenn@u.washington.edu (B.K.K.)

Calorie restriction (CR), instituted by reducing the glucose concentration of the media from 2% to 0.5% or lower, increases yeast replicative life span and was proposed to work through a Sir2-dependent mechanism (1). We recently reported that CR increases life span in the long-lived BY4742 strain to a greater extent in cells lacking Sir2 than in wild-type cells, as long as extrachromosomal ribosomal DNA (rDNA) circles are kept at low levels by deletion of the gene coding for the replication fork block protein, Fob1 (2). This discovery has since been confirmed in a report by Lamming et al. (3), in which they extended our work and reported that CR fails to increase the life span of cells lacking Sir2, Hst2, and Hst1. Based on this finding, Lamming et al. (3) propose that Sir2-family proteins (sirtuins) are activated by CR, and a Sir2-redundant function of Hst2 (and to a lesser extent Hst1) as a repressor of rDNA recombination accounts for Sir2-independent life-span extension by CR.

To test the model of Lamming et al. (3), we generated yeast lacking Sir2, Hst1, Hst2, and Fob1 and determined the effect of CR on life span. Contradictory to the results of Lamming et al. (3), we observed a significant life span extension at 0.5% (23% increase in mean life span, P = 0.003), 0.05% (66% increase in mean life span, P = 1.3 x 10–8), and 0.005% (72% increase in mean life span, P = 4.9 x 10–9) glucose in BY4742 sir2{Delta} hst1{Delta} hst2{Delta} fob1{Delta} mother cells (Fig. 1A). Lamming et al. (3) also report that a genetic model of CR, deletion of the gene encoding hexokinase, HXK2, fails to increase the life span of BY4742 sir2{Delta} fob1{Delta} hst1{Delta} hst2{Delta} mother cells. In contrast, we found that sir2{Delta} fob1{Delta} hst1{Delta} hst2{Delta} hxk2{Delta} mother cells were significantly longer lived (62% increase in mean life span, P = 6.3 x 10–7) than sir2{Delta} fob1{Delta} hst1{Delta} hst2{Delta} mother cells (Fig. 1B). These data are consistent with the model that Sir2-independent life-span extension by CR is not mediated by Hst2 or Hst1 (or both).


Figure 1  Fig. 1. Life-span extension by CR occurs independently of Sir2, Hst1, and Hst2. (A) CR significantly increases the life span of BY4742 sir2{Delta} fob1{Delta} hst1{Delta} hst2{Delta} mother cells at 0.5%, 0.05%, and 0.005% glucose. (B) Deletion of HXK2 significantly increases the life span of BY4742 sir2{Delta} fob1{Delta} hst1{Delta} hst2{Delta} mother cells. © CR fails to increase the life span of W303AR5 mother cells. Mean life spans shown in parentheses. [View Larger Version of this Image (17K GIF file)]


In addition to BY4742, the W303AR5 strain was used by Lamming et al. (3) to examine Sir2-independent life-span extension by CR. In previous work from the Sinclair and Guarente labs (4–6), experiments measuring the effect on life span of CR by growth on low glucose have been carried out solely in the PSY316 strain background, with W303AR5 used for rDNA recombination analysis and life span experiments involving SIR2 or FOB1. To the best of our knowledge, the report by Lamming et al. (3) is the first to claim life-span extension from growth on reduced glucose in W303AR5. We therefore further examined the effect of CR in W303AR by determining the life span of W303AR5 cells at different glucose concentrations ranging from 2% to 0.05%. We found no significant life-span extension by CR at any reduced glucose level (Fig. 1C). It is unclear why the data of Lamming et al. (3) differ from ours. All of our experiments were carried out using the standard yeast replicative life-span methodology (2, 7, 8), with researchers performing the micro-dissection blind to the identity of individual strains within each experiment.

In addition to our findings reported here, the notion that Sir2 and other sirtuins redundantly mediate the CR response, as proposed by Lamming et al. (3), is difficult to reconcile with several observations. First, CR increases life span by a greater magnitude in fob1{Delta} cells relative to wild-type cells, irrespective of Sir2, Hst1, and/or Hst2 activity (2). Second, PSY316, a strain that shows robust life-span extension in response to CR, shows no life-span effect in response to increased expression of Sir2 (9). Third, the in vivo activity of Sir2, as measured by telomere silencing, is not enhanced by CR (7, 8). Finally, CR does not extend life span in a sir2{Delta} FOB1 strain (1), and Lamming et al. (3) offer no explanation as to why CR does not activate Hst2 and Hst1 to offset the loss of Sir2 under these conditions. Further, the report by Lamming et al. (3) that Hst2 inhibits rDNA recombination in a Sir2-independent fashion contradicts previous findings by Gasser's group (10).

It is important to resolve the controversy over whether sirtuins function to mediate life-span extension in response to CR. Parallel studies are ongoing in other model systems such as Caenorhabditis elegans, Drosophila melanogaster, and mice. Our data here demonstrate that Sir2, Hst1, and Hst2 are not required either alone or in combination for life-span extension by CR in yeast, consistent with the model that CR is not mediated by sirtuins in this organism.

References and Notes

    * 1. S. J. Lin, P. A. Defossez, L. Guarente, Science 289, 2126 (2000).[Abstract/Free Full Text]
    * 2. M. Kaeberlein, K. T. Kirkland, S. Fields, B. K. Kennedy, PLoS Biol. 2, E296 (2004). [Medline]
    * 3. D. W. Lamming et al., Science 309, 1861 (2005); published online 28 July 2005 (10.1126/science.1113611).[Abstract/Free Full Text]
    * 4. R. M. Anderson, K. J. Bitterman, J. G. Wood, O. Medvedik, D. A. Sinclair, Nature 423, 181 (2003). [CrossRef] [ISI] [Medline]
    * 5. M. Kaeberlein, M. McVey, L. Guarente, Genes Dev. 13, 2570 (1999).[Abstract/Free Full Text]
    * 6. S. J. Lin et al., Nature 418, 344 (2002). [CrossRef] [ISI] [Medline]
    * 7. M. Kaeberlein et al., Science 310, 1193 (2005).[Abstract/Free Full Text]
    * 8. M. Kaeberlein et al., PLoS Genet. 1, e69 (2005). [CrossRef] [Medline]
    * 9. M. Kaeberlein et al., J. Biol. Chem. 280, 17038 (2005).[Abstract/Free Full Text]
    * 10. S. Perrod et al., EMBO J. 20, 197 (2001). [CrossRef] [ISI] [Medline]

Response by researchers

Response to Comment on "HST2 Mediates SIR2-Independent Life-Span Extension by Calorie Restriction"
Dudley W. Lamming,1 Magda Latorre-Esteves,1 Oliver Medvedik,1 Stacy N. Wong,2 Felicia A. Tsang,2 Chen Wang,2 Su-Ju Lin,2* David A. Sinclair1*

Our two labs and others have shown that SIR2 controls the life span of diverse species, including Saccharomyces cerevisiae and Drosophila melanogaster, and that deleting SIR2 blocks life-span extension by calorie restriction. The methods of Kaeberlein et al. allow yeast to bypass the requirement for SIR2 and its homologs, which brings into question their suitability for modeling the physiology of more complex organisms.

1 Paul F. Glenn Laboratories, Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
2 Center for Genetics and Development and Section of Microbiology, University of California, Davis, 351 Briggs Hall, Davis, CA 95616, USA.

* To whom correspondence should be addressed. E-mail: slin@ucdavis.edu (S.-J.L.); david_sinclair@hms.harvard.edu (D.A.S.)

We believe it is important to understand why experiments performed by Kaeberlein et al. (1, 2) allow yeast to bypass the requirement for SIR2, HST1, and HST2 during calorie restriction (CR) and how relevant this observation is to complex organisms. The simplest explanation is that there are at least two pathways for life-span extension in Saccharomyces cerevisiae: a sirtuin-dependent pathway and a sirtuin-independent pathway, the latter of which is invoked by the methods of Kaeberlein et al., which induce more intense nutrient restriction.

The life-span extensions shown by Kaeberlein et al. (1) were obtained using a protocol that differs substantially from ours (3–5). Although the authors state that reducing glucose by 97.5% is standard methodology for inducing CR, we are unaware of any other lab that uses this protocol. While we can debate the merits of reducing glucose to this extent, and have done so previously (5, 6), the fact that Kaeberlein et al. do not observe sirtuin-dependent effects of CR suggests that their methods may be unsuitable for understanding the role sirtuins play in mediating CR in more complex organisms (7, 8).

In our report (9), we performed the key experiments in at least two yeast strains, and data from our two laboratories were, and continue to be, consistent. In contradiction to Kaeberlein et al., we find that 0.5% glucose produces a negligible life-span extension in BY4742 sir2{Delta} fob1{Delta} hst1{Delta} hst2{Delta} (Fig. 1A). A clue as to why the life-span data of Kaeberlein et al. contradict not only our two labs but also data from Guarente and Goldfarb (3, 10) may be found in the observation that W303 does not live longer when subjected to severe nutrient limitation (Fig. 1B). The inability of Kaeberlein et al. to observe CR-mediated life-span extension in W303 indicates that their protocol induces greater nutrient stress on cells than does ours. Another clue is that even on 2% glucose medium, BY4742 cells live an average of 13 to 20% longer in the hands of Kaeberlein et al. than in ours [compare our Fig. 1A with figure 1, A and B, in (1)], which indicates that their protocol induces a greater degree of nutrient stress and induces longevity pathways even on 2% glucose.


Figure 1  Fig. 1. Effects of CR on yeast life span and rDNA recombination. (A) Calorie restriction (0.5% glucose) does not extend life span in BY4742 sir2{Delta} fob1{Delta} hst2{Delta} hst1{Delta} strain, one-tenth of that glucose concentration does. Life-span analyses were performed as previously described (8). Average life span: 2% glucose, 22.4; 0.5% glucose, 21.1; 0.05% glucose, 27.5. (B) W303 responds to CR but is sensitive to more intense glucose restriction. Average life spans: W303AR5 2% (w/v) glucose, 23.9; 0.1% glucose, 24.9; W303AR5 sir2{Delta} fob1{Delta} 2% glucose, 26.1; 0.1% glucose, 28.0. © Sir2 and Hst2 are recruited to the rDNA during CR (16). (D) CR suppresses rDNA recombination in the absence of SIR2. Ribosomal rDNA recombination rates were determined as previously described using a half-sector–based colony assay (8). Cells were maintained in log phase for 8 hours in the indicated medium before plating to YPD medium. [View Larger Version of this Image (36K GIF file)]


We thought it self-evident that to make assertions about the effect of CR on ribosomal DNA (rDNA) recombination, one would need to measure it. Despite the authors' strong claims about a variety of genes being relevant or irrelevant to rDNA recombination (11), they do not measure either rDNA recombination or extrachromosomal rDNA circles (ERCs). Measures of telomeric silencing cited by Kaeberlein et al. (1) are misleading because of known locus-specific differences in how Sir2 activity is regulated (12) and the increasing number of examples where silencing of a marker gene at the rDNA does not correlate with recombination (13, 14). By measuring rDNA recombination, we consistently observe that it is reduced by half in response to 0.5% glucose, and that this reduction is abrogated by deleting SIR2 and HST2 and completely eliminated by nicotinamide, a sirtuin inhibitor (9). Moreover, there is a twofold increase in the abundance of Sir2 at the NTS2 region of the rDNA during CR, and in the absence of Sir2, Hst2 is recruited there (Fig. 1C), consistent with our model (9).

Kaeberlein et al. ask why Hst1 and Hst2 do not offset a loss of SIR2. Loss of SIR2 results in hyper-recombination, which is countered by Hst1 and Hst2 during CR, but not to a level that extends life span significantly (Fig. 1D). When both SIR2 and FOB1 are deleted, thus lowering the amount of rDNA recombination to near wild-type levels, the effects of Hst1 and Hst2 are more apparent. As we stated, Hst1 and Hst2 are thought to augment the activity of Sir2 during CR, and their activity is more evident when SIR2 and FOB1 are deleted.

Kaeberlein et al. state that our work contradicts the work of the Gasser lab (15), but the two studies are not directly comparable. The Gasser lab did not study CR, did not measure the effects of HST2 on life span, and used a less sensitive assay for measuring rDNA recombination.

Lastly, Kaeberlein et al. have found conditions under which life span can be extended by CR in the absence of sirtuins and have concluded that they play no role. However, it is an accepted rule of genetics that if one finds that a phenotype can occur in the absence of a gene, it does not mean that said gene plays no role. A more apt interpretation is that there is an alternative pathway that can be activated under certain conditions. We look forward to future discoveries about the complexities and redundancies that underlie life-span extension by CR in yeast and multicellular organisms.


References and Notes

    * 1. M. Kaeberlein et al., Science 312, 1312 (2006); www.sciencemag.org/cgi/content/full/312/5778/1312b.
    * 2. M. Kaeberlein et al., PloS Biol. 2, 296 (2004). [CrossRef]
    * 3. R. M. Anderson et al., Nature 423, 181 (2003). [CrossRef] [ISI] [Medline]
    * 4. S. J. Lin, P. A. Defossez, L. Guarente, Science 289, 2126 (2000).[Abstract/Free Full Text]
    * 5. S. J. Lin, L. Guarente, PLoS Genet. 2, e33 (2006). [CrossRef] [Medline]
    * 6. D. A. Sinclair, S. J. Lin, L. Guarente, Science 312, 195d (2006).[Free Full Text]
    * 7. B. Rogina, S. L. Helfand, Proc. Natl. Acad. Sci. U.S.A. 101, 15998 (2004).[Abstract/Free Full Text]
    * 8. D. Chen, A. D. Steele, S. Lindquist, L. Guarente, Science 310, 1641 (2005).[Abstract/Free Full Text]
    * 9. D. W. Lamming et al., Science 309, 1861 (2005).[Abstract/Free Full Text]
    * 10. S. Jarolim et al., FEMS Yeast Res 5, 169 (2004). [CrossRef] [ISI] [Medline]
    * 11. M. Kaeberlein et al., Science 310, 1193 (2005).[Abstract/Free Full Text]
    * 12. J. C. Tanny, D. S. Kirkpatrick, S. A. Gerber, S. P. Gygi, D. Moazed, Mol. Cell. Biol. 24, 6931 (2004).[Abstract/Free Full Text]
    * 13. K. T. Howitz et al., Nature 425, 191 (2003). [CrossRef] [ISI] [Medline]
    * 14. J. Huang, D. Moazed, Genes Dev. 17, 2162 (2003).[Abstract/Free Full Text]
    * 15. S. Perrod et al., EMBO J. 20, 197 (2001). [CrossRef] [ISI] [Medline]
    * 16. Chromatin immunoprecipitation was performed from extracts of W303AR5 yeast in which the endogenous SIR2 or HST2 gene was modified to express a C-terminal 3 x HA tag. Cultures were grown to an OD600 = 1.0 in 2% or 0.5% (CR) glucose and then cross-linked for 15 min at room temperature using 2% formaldehyde. Immunoprecipitation was performed using a rabbit polyclonal antibody to HA (Abcam). Primer sequences used for amplifying DNA at NTS2 and ACT1 have been previously published by Huang et al. (14); primer set 23 was used. Relative enrichment in the immunoprecipitated sample for NTS2 versus ACT1 was determined using quantitative PCR.

[knowitall]

I think David Sinclair is going for some kind of record for the most Science and Nature papers that are bogus