Substrate specific activation fo sirtuins by resveratrol.

[Jason]

Well, i am a little late to the party, but a few comments on this
thread (google won't let me post a reply to the original):
"Fundamental laboratory error" is an overstatement, and claims that the
whole thing was made up are simply false. Nor is the work "faulty."
The test used was a well characterized assay for deacetylase activity,
and the interpretation of the results was consistent with the data
generated. In short, here's the scoop--the fluorescent moiety attached
to the peptide turned out to decrease the binding affinity of the
peptide for SIRT1. Resveratrol binds SIRT1 and then increases the
affinity for this lower-affinity substrate. This increase is not seen
in a fluorescent-less peptide, which binds much tighter. We have also
observed that the results of these assays can vary depending on the
concentration of substrate and NAD that are used. If the starting
concentration is too high, even with the fluorescent peptide, the
effect goes away. Resveratrol binding to SIRT1 is gaining acceptance in
the scientific literature. I would point you to an excellent recent
paper in JBC from John Denu's lab (available free on pubmed) examining
the biochemistry in more detail. They find similarly that activation in
the assay depends on the fluorescent moiety, but note they agree that
resveratrol _can_ bind SIRT1 and change substrate affinity. The real
question is how tight are the actual proteins binding? (under
investigation) If in vivo targets bind with a Kd more akin to the
labelled peptide, rather than the unlabelled, one could easily imagine
how the effect might be physiologically relevant.
As far as the lack of reproducibility of in vivo yeast data--that is
puzzling I agree, especially since the authors of the study are among
the world's leaders in performing yeast lifespans. I will only say at
least one other lab has repeated the yeast results, several labs have
repeated the worm results (with sir2 dependence), and flies have been
confirmed in at least two labs (with sir2 dependence).
So yes, it is not as straight forward as we had hoped. No, we didn't
make the whole thing up, and the sky is not falling.
Armchair scientists should be careful in drawing conclusions and
flinging accusations when they do not have first hand knowledge of
what's going on.
Sincerely,
Jason Wood

tcar...@elp.rr.com wrote:
> Hi rs1,
> This could be a very important paper. It appears that the
team
> of Sinclair DA has made a fundamental laboratory error. Perhaps that
of
> Guerente also, my memory is not that good on his work. At any rate
both
> have founded well financed startups which apparently will try to
market
> some resveratrol cocktail based in part on their faulty work which
may
> now be exposed. A list I posted a while back which gave small
molecule
> activators of SIR2 is also now of dubious value.
> Will the resveratrol bubble now burst? The presumed
activation
> of the SIRT family was a corner stone of the rapidly accumulating in
> vitro and non mammalian eukaryote evidence altho much independent
> evidence remains.
> Sinclair's mistake, of course, was that his probe, fluor de
> lys, apparently affected the molecules under study. If it was truely
a
> mistake hopefully others will be more conscientous with their work.
> One of Sinclair's co-authors, Jason Wood was kind enought to
> make several posts here concerning their work. If he's still reading
> perhaps he will comment.
>
> Thomas

[tcar...@elp.rr.com]

Hi Jason,
Thx for replying. I think most of us, certainly I for one
believe your team is on the very short list for being the best
resveratrol researchers in existence despite the unfortunate flur de
lys experiment, and we are routing very strongly for you. If you
manage to put together a good cocktail we will be among your first
customers. Regardless of its relationship with sirtuins resveratrol is
widely thought to be a beneficial substance. I as well as others of
this group now supplement with it hoping that the issue of
bioavailability falls our way. With no human studies the guinea pig
study I attach below is certainly encouraging.
I was hoping you would take issue with my report and tried
to make it accurate, but not unnecessarily critical. I guess I need
more practice. Now that I have the attention of a world class scientist
I can't resist making another critical observation that I made some
time ago. I think that when substances which are strongly conjugated by
the human gut or liver are studied in vitro, physiologically
appropriate mixtures of the aglycones and conjugates should be used. In
the case of resveratrol the glucuronides and sulphates that are present
in human plasma would be the substances that should dominate the
mixture in the same percentage seen in our plasma. IMO This seems so
obvious to me that I know there must be some reasons it's not done.
Perhaps you might be able to shed some light on this.

Thomas

Oral administration of trans-resveratrol to guinea pigs incr­eases
cardiac DT-diaphorase and catalase activities, and protects ­isolated
atria from menadione toxicity.
Floreani M, Napoli E, Quintieri L, Palatini P.
Department of Pharmacology and Anesthesiology, University of­ Padova,
Largo Meneghetti 2, 35131, Padova, Italy. maura.flore...@uni­pd.it
<maura.flore...@unipd.it>
Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a
natural ­phytoalexin
found in grapes and wine. It has antioxidant and antiprolife­rative
activities, and has been shown to induce NAD(P)H:quinone
oxidoreductase, also known as DT-diaphorase, in cultured mou­se
hepatoma
cells. DT-diaphorase is a detoxifying enzyme for quinone-con­taining
substances, due to its ability to prevent their one-electron­
reduction
and the consequent generation of reactive oxygen species (RO­S). The
aim
of the present study was to investigate whether oral adminis­tration
of
trans-resveratrol to guinea pigs (60 mg/l in tap water for 1­6 days,
ad
libitum) increases cardiac DT-diaphorase and, consequently, ­reduces
the
response of isolated atria to 2-methyl-1,4-naphthoquinone
(m­enadione),
the positive inotropic effect of which is related to the amo­unt of
ROS
generated by its cardiac metabolism. In the cardiac tissue o­f
resveratrol-treated animals, DT-diaphorase activity was sign­ificantly

higher than that measured in control animals, the V(max) of ­the
enzyme
reaction being 75.47 +/- 3.87 and 50.73 +/- 0.63 nmoles/mg
p­rotein/min,
respectively (p < 0.05). Resveratrol administration also
sig­nificantly
increased the activity of cardiac catalase (32.20 +/- 2.39 v­s. 25.14
+/- 3.85 units/mg protein in treated and control animals,
re­spectively;
p < 0.001). As a consequence, menadione metabolism by the ca­rdiac
homogenate obtained from resveratrol-treated animals generat­ed a
smaller amount of ROS and, in electrically driven left atria­,
menadione
produced a significantly lower increase in the force of cont­raction
than in atria isolated from control animals. These results i­ndicate
that oral administration of resveratrol exerts cardioprotect­ion
against
ROS-mediated menadione toxicity. PMID: 12679191
Posted by Tim and it's probably the best argument that
resveratrol supplementation does indeed have effects in humans despite
poor bioavailability.

[Jason]

Thomas,
my vitriol was directed primarily at others who were harsher than you.
You raise valid questions. I agree with you that resveratrol seems to
have a lot of great properties, and a good many of the effects are
likely to have little to do with sirtuins.
With regard to conjugates--
you are entirely correct. These should be researched in the in vitro
assays. I have heard rumors that this is currently being done. One
thing to remember is the angle that we approached this problem from. We
are not pharmacologists, and are primarily interested in answering
questions about the basic biology of sirtuins and how they relate to
aging in our simple model systems from a genetic and biochemical
direction. Flies, worms, and yeast do not have livers. It seemed to
make sense to begin in vivo testing with the compound that came out of
the screen, which was the unconjugated resveratrol. Science in the lab
frequently does not develop in quite the orderly way media reports or
even finished published papers project. That having been said, results
using the conjugates you mention would certainly be exciting from the
point of view of (and perhaps more relevant to) human biology, which is
what we all ultimately care about. I, with you, look forward to seeing
more data.
JW

[tcar...@elp.rr.com]

Hi Jason,

Fine answer. Best wishes in your endevor.

Thomas