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[A1CR]

The following message from 2004-01-17 was posted on behalf of MR. Here MR comments on the potential downside of most of the reservatrol supplements currently on the market. According to MR, virtually all of them are the wrong form, and may very well be harmful, rather than beneficial:

There's been much interest of late in the possibility that "resveratrol"
(a polyphenl found in grapes and red wines) may be a CR-mimetic.
Trans-resveratrol certainly has such effects in yeast (a 70% increase in
replicative LS (1)), & it's related to activation of Sir2, the yeast
sirtuin histone deacetylase, which is certainly a "CR gene" in these
critters. I've often expressed skepticism about the relevance of the
yeast CR-Sir2 work in the past [KH: search the Archives at calorierestriction.org for more info]

... but I've become cautiously optimistic of late, because:

1. Genetic & other interventions that increase Sir2 levels or activities have
now been shown to extend LS in multicellular organisms (flatworms,
fruit flies (5));

2. CR's life extension & sirtuin-boosting activity in yeast happens
INDEPENDENTLY of NAD levels, which latter, in fact, go DOWN on CR: "CR
reduces nuclear NAD(+)levels in vivo. Moreover, the activity of Sir2 and
its human homologue SIRT1 are not affected by physiological alterations
in the NAD(+):NADH ratio. These data implicate alternate mechanisms of
Sir2 regulation by CR" (2) -- alternate, that is, to the previous
hypothesis linking NAD, sirtuin, and CR, under which CR, by reducing the
flow of electrons from food energy thru' to the mitochondria, would free
up more NAD because less NADH would be formed.

The problem with this hypothesis is that, even if it were true in yeast,
it's contrary to the bulk of the research on CR & metabolic rate in
MAMMALS, which shows that (despite the still-widely-held "common sense"
belief to the contrary) CR in mammals does NOT reduce specific metabolic
rate: after a transient depression, SMR bounces right back. (This is not
a fully-resolved issue, however, for a variety of reasons that I won't
get into now).

So, based on current data, there would be no predicted "freeing up" of
NAD in CR mammals, & no resulting facilitation of sirtuin activity -- in
which case all of the yeast, worm, and fly CR data related to sirtuins
would be irrelevant to humans. But if CR induces sirtuin activity in
yeast INDEPENDENTLY of NAD (2), then that eliminates this theoretical
objection, making the possibility that sirtuin activation is a/the
mechanism of CR in mammals a more plausible hypothesis -- & hence,
increasing the likelihood that "SirTuin-Activating Compounds" (STACs) might
really work as CR-mimetics.

3. There is now epidemiological evidence for the importance of sirtuins
in humans. Men with a more favorable variant f an human sirtuin have a
longer life expectancy at any given age than the population at large, &
those with a less favorable variant, a shorter one (3).

All of this makes me more bullish (but, NB, far from CONVINCED) than
I've been a few months ago about a molecule that fires up sirtuins &
extends replicative LS in yeast. This is all the more so granted that
the trans-resveratrol results in yeast also apparently apply to simple
multicellular organisms: "preliminary experiments indicate that
[trans-]resveratrol can also extend the lifespan of multicellular
animals, including the nematode C. elegans and the fruitfly Drosophila
melanogaster (J.G.W., D.A.S. and M. Tatar, unpublished results)." (1) In
a radio interview on NPR (4), Sinclair specified "something more like a
50% increase" in LS in the latter.

Accordingly, there's much interest in resveratrol supplements. With all
of the [...] interest & propaganda from Life Enhancement Products,
LEF, and other supplement companies, I've been getting a lot of [...]
questions about resveratrol supplements in general. What
I've been telling folks, in brief, is: "Do not take any currently
commercially-available "resveratrol" supplements; I'll explain shortly."

This took longer than 'shortly;' amusingly, the Longevinex press release [KH: search the Archives at calorierestriction.org for more info]
... & website < http://www.longevinex.com > appeared in the interim,
confirming my suspicion that extant "resveratrol" supplements don't do the job. Aplogies to all for the delay; my rationale
-- and a plug -- follows.

To cut to the chase, the issue is the fact that all "resveratrol"
supplements presently on the market -- except,
now, Longevinex -- contain little of their "resveratrol" in the *trans*
configuration, and much of this is in a glucoside form of uncertain
pharmacokinetics. Nearly all supplements come from Japanese knotweed
(Polygonum cuspidatum), and ~3/4 of the total "resveratrol" is either
cis-resveratrol & its glucosides or trans-resveratrol *glucosides*.

This is unlike the case with grapes, which synthesize only the
trans-isomer: the enzyme that produces it, stilbene synthase, is
specific for the trans-configuration, & the cis-form is produced when
trans-resveratrol is isomerized by exposure to ultraviolet & heat (11).
Grapes contain no (6-9a) or unquantifiably low (10) levels of the
cis-isomer; wine contains some, but the bulk is still in trans-orientation.

The presence of high levels of the cis-isoforms in extant "resveratrol"
supplements could potentially be a very bad thing -- ESP for practicing
CR folk.

Sinclair et al originally chose to try trans-resveratrol per se, based
precisely on its structure, after finding that the most effective
previously-tested polyphenols shared a couple of key structural features
-- in particular, the trans- isomerization. "The initial compound screen
identified ... two structurally similar compounds, quercetin and
piceatannol, that stimulated SIRT1 activity five- and eightfold,
respectively ..."

"Comparison of the two compounds suggested a possible structure–
activity relationship. The trans-stilbene ring structures of piceatannol
are superimposable on the flavonoid A and B rings of quercetin ...
Furthermore, the 5, 7, 3’ and 4' hydroxyl group positions in quercetin
can be aligned, respectively, with the 3, 5, 3' and 4' hydroxyls of
piceatannol. … A secondary screen of these compound families identified
15 additional [human sirtuin] activators that provided more than twofold
stimulation … Many, but not all of the most active STACs included
hydroxyls in the two meta-positions of the A ring, trans to the B ring
with a 4' or 3',4' hydroxyl pattern … A potentially coplanar orientation
and trans-positioning of the hydroxylated rings also appear to be
important for activity ... The most potent activator was resveratrol
(3,5,4'-trihydroxystilbene) ..." (1)

Interestingly, the SAME structural characteristics are apparently
obligatory for key non-antioxidant anticancer mechanisms of
trans-resveratrol: "the presence of 4'-OH together with stereoisomery in
the trans-conformation (4'-hydroxystyryl moiety) was absolutely required
for inhibition of cell proliferation. Enzymatic assays in vitro
demonstrated that inhibition of DNA synthesis was induced by a direct
interaction of resveratrol with DNA polymerases" (18).

Now, this means that, AT BEST, cis-resveratrol is likely to be inactive.
But I suggest that there is reason to suggest that they are not merely
inactive, but COUNTERACT the effect of the trans-isomer: that
cis-resveratrol in particular is, in effect, a "Sirtuin Inactivating
Compound" (SIC -- sic !). By hook or by crook, the "other"
"resveratrols" in standard "resveratrol" supplements are, I hypothesize,
the reason for the fact that nearly all such supplements did not
increase sirtuin activity in the assay reported by Sinclair's corporate
partners on their website: < http:/ www.longevinex.com > (click forward
to second screen).

While we don't know the details of the assay, I assume that they likely
did was basically what was done in (1): take either human cells or (mre
likely) recombinant human SIRT1, and applied the product at a
concentration such that its TOTAL "resveratrol" was up at the
concentration previously determined (in (1)) to maximally boost sirtuin
activity. Alternatively, they may have actually put in enough to bring
just the trans-isomer (which would lead to proportionately greater still
concentrations of the other isomers).

It would be predicted, from the structure/activity relationship outlined
above, that at the very least the cis-resveratrol would be inactive. But
if that were the case, one would predict SOME sirtuin activation by
these products, proportional to the amount of the free trans-isomer
whcih they contain -- say, 25% of that recorded for Longivinex -- &
more, under the latter scenario. Instead, for all but 2 products, NO
activation was measured.

Now, the Longevinex website -- apparently as a mouthpiece for Sinclair
-- suggests that this is due to oxidation of the resveratrol. In my
usual arrogant fashion, I'll just state that this is nonsense. One can
easily measure the degree of degradation in a resveratrol supplement;
the result usually shows a difference which is within the experimental
error. Unstable as it may be in wine, the stuff is quite stable in
dry extract form sitting in a capsule in a bottle for a couple of
years; likewise, vitamin C is rapidly depleted out of orange juice, but
vitamin C tablets remain potent for a long, long time. The small losses
of "resveratrol" in these products certainly would not account for ZERO activity.

Shortly after the epub of Sinclair's paper (ie, long before seeing the
Longevinex website report), I formulated a SPECULATIVE counterhypothesis:
those other "resveratrol" molecules are not just inactive, but are
actually COUNTERPRODUCTIVE -- on the fact that just such effects are
common with opposing isomers of biomolecules which act on/with enzymes
in vivo. The examples with which many folks on this List will be
familiar will include S(-)-lipoic acid (which INHIBITS the effects of
the orthomolecular R(+)-enantiomer on mitochondrial functin and insulin
signaling -- see the "Discussion" section of (12), and extensive
(commercial) discussion at < http://www.R-Lipoic.com >), and also
D-carnitine, whch opposes the action of L-carnitine such that, whereas
people with angina-related exercise intolerance experience improvements
in peak V02, lactate threshold and ventilatory threshold after using
pure L-carnitine supplements, the same parameters WORSEN after use of
DL-carnitine (17). When administered to folks needing dialysis (& hence
poor clearance of the stuff), the racemic carnitine mixture actually
induces MYASTHENIA in a significant % of patients (13-16).

This would seem an esp apt argument if the "xenohormesis" hypothesis of
Sinclair et al (that critters that eat plants have adapted via evolution
to sense the presence of trans-resveratrol and other phytoalexins
(environmental stressor response molecules) in their foods as
bellweathers for coming food shortages by inducing a CR response) were
true: trans-resveratrol would not just be a pharmacological substance,
but a borderline orthomolecule to which the organism were specifically adapted.

(The obvious counterexample is d,l-alpha-tocopherol, which is merely
less well-retained and active in the tissues than pure d-alpha. This is
actually a non-exception, however, as there is no actual coenzymatic
function of alpha-tocopherol, & hence no enzyme for it to bugger up).

Now, if you expose cells or recombinant SIRT1 to pure trans-resveratrol,
you see activation of the protein. If the cis-isomer were merely
inactive, you'd see a degree of activation proportional to the
concentration of the trans-isomer. The fact that nearly all of the
tested supplements demonstrated NO net activity is very consistent with
my hypothesis: the cis-isomer is counteracting the trans-, leading to no
net effect.

And in fact, though they aren't mentioning it, it seems that
Sinclair/Longevinex also consider this to be important. While the
"Supplement Facts" box on the product looks similar to regular
"resveratrol" products, & even seems to've derived most of its
"resveratrol" from Polygonum:

http://www.longevinex.com/sdm.asp?pg=products&specific=jnnmmpe0

"Proprietary Longevinex (tm) blend 100 mg
Providing not less than 15 mg of resveratrol, from Giant knotweed
(Polygonum cuspidatum) leaf extract
Red Grape (Vitas vinifera), skin extract
--------
Quercetin, 75 mg
Phytic acid 25 mg
Lecithin 100 mg"

... none the less, when I called, the company's rep assured me that in
fact it is overwhelmingly free trans-resveratrol, with only trace
amounts of the cis-. This CAN be done -- & not only in principle,
either: over the course of composing this monster post, I've actually
discovered, in the existing literature, a quite effective way of doing
this, which is so simple as to be really stovetop chemistry. (No, I
won't tell ). That would both suggest that "they" (Sinclair?
Longevinex?) consider the purging of the cis-isomer to be important, &
explain why Longevinex, like Sinclair's lab material (reagent-grade
trans-resveratrol) shows powerful activity.

The presence of the trans-resveratrol *glucosides* in standard
"resveratrol" supplements complicates matters. These are typically the
plurality of the total "resveratrol" in a supplement. I would PRESUME
that they would be inactive in this in vitro assay, but (as is typically
seen with glycosylated flavonoids) that the glucose moiety woud be
cleaved prior to entry into the circulation. This might be expected to
be a GOOD thing -- suddenly, in vivo, there's much more active trans-
isomer available than would be seen in the in vitro assay, perhaps
sufficient to counteract the negative impact of the cis- -- but in fact
it's not at all clear to me that this would be so, because glycosylation
of flavonoids also has strong impact on their bioavailability.

Unfortunately, all the pharmacokinetic data I've seen (discussed below,
in the context of dosing) is on straight reagent-grade
trans-resveratrol. But certainly there are cases of glycosylated
flavonoids with both increased and decreased bioavailability relative to
the free form. Eg. isoflavones are much more bioavailable in their
aglycone form, while quercetin is significantly more bioavailable in the
form of a specific glucoside. The main mechanism of action of quercetin
absorption is not passive diffusion, but an active process which
exploits the Na-dependent glucose transporter SGLT1, following which the
glucose moiety is cleaved off in the intestinal cell by
beta-glucosidases, and then the free (or subsequently Phase-II conjugated) quercetin molecule
passes on into the circulation. As a reslt, the glycosylated
quercetin-3-O-glucoside is significantly better-absorbed than the free
form ((19-26) on all of this).

If resveratrol is more like quercetin than soy isoflavones (as seems
most likely, due to the close structural resemblance of the 2 --
resemblance so close as to trigger the investigation of trans-rseveratol
in the first place, after the initial, weaker findings re: q itself as a
STAC), then one might expect a significant increase in its
bioavailability. This sounds, initially, in the "more-is-better"
mentality of the supplement-sucking universe, like a good thing -- until
you remember that (1) specifically reports that whereas "Treatment of
cells with a low concentration (0.5micM) of [trans-]resveratrol
increased cell survival after ionizing irradiation" and "U2OS cells
treated with 0.5micM resveratrol showed a marked decrease (about 75%) in
the level of Ac-K382" -- indicating activation of deacetylation activity
by SIRT1 -- "At higher concentrations of [trans-]resveratrol >50micM)
**the effect was reversed,** which may explain the dichotomy in the
literature regarding the effects of resveratrol on cell viability" (1).

Now, if you take straight trans-resveratrol, we can be reasonably
confident of the amount required to achieve plasma levels within the
golden zone. In (27), volunteers "were randomly assigned to three
different groups consuming orally ... trans-resveratrol, 25 mg/70 kg ...
randomly administered at 4-week intervals in three different matrices
... [P]olyphenols were present in serum and urine predominantly as
glucuronide and sulfate conjugates ... The free polyphenols accounted
for 1.7 to 1.9% (trans-resveratrol)".

The actual pharmacokinetics are illustrated in the Figures. From Figure
2, TOTAL resveratrol metabolites peak at about 30 mins, at ~450 mcg/L,
which (granted a molecular weight of 228.25) is not MORE than 2.19
micromoles/L, well below the 50 micmol/L reversal point, & is in fact
less, because the conjugated metabolites will have a higher weight than
the free compound. In terms of FREE trans-resveratrol per se (the only
one likely to be active, & certainly the only one with evidence to
suggest such (as the authors of (27) pessimistically (they think!)
note), Figure 5 shows that the peak is 7-8 mcg/L, or ~0.03 mcmol/L --
suggesting that one would want twice this much (50 mg trans-resveratrol)
to achieve target plasma levels.

However, these are PEAK levels. T-r levels were significantly down at
the 1 h amark, & were down to essentially baseline levels at 2 h (with
the conjugatesshowing a somewhat longer life in the body). This might
mean taking 50 mg or more every 1-2 h throughout the day, unless there is
a "memory effect" on the sirtuins due eg. to either a sustained shift in
cellular metabolism or to the molecule itself hanging around behind the
cell membrane for longer -- but in the latter case it's also likely
they'd be biotransformed intracellularly, & on a tissue-specific basis
at that ...

We don't have such data for the glucoside. As a result, it's quite
possible that the presence of large amounts of same might lead one out
of the golden zone via a (currently quite unpredictable) free
trans-resveratrol "overdose." In the absence of a good way to predict
the pharmacokinetics, I suggest that this is just a molecule to avoid
in supra-dietary quantities.

The standard "resveratrol" supplement, IOW, contains molecules likely on
theoretical and empirical grounds alike at best cancel out the STAC
activity of the trans-resveratrol they contain. In an ALer, this might
just render such supplements useless. But in a CR practitioner, it could
be disastrous -- IF (still a big 'if,' NB) sirtuin activation is indeed
important to the CR effect. Reason: the cis-resveratrol, in particular,
might DOWNregulate a CR-induced sirtuin activation -- as might the
glucosides, if one took enough of the stuff (at present, of course, no
one does, if they're taking a standard <=500 mg, 8% "resveratrol"
supplement).

What one wants, then, is a pure trans-resveratrol supplement, of
pharmaceutical grade (not reagent (which latter is already available
from Sigma-Aldrich, etc)), at a minimum dose which might reasonably be
placed at ~50 mg. No such supplement presently exists: they're either
isomerically impure, or provide the stuff at an insufficient dose.

[...]

Just to be *extra-sure* that we keep the stuff stable, any such
supplement would be put in PET bottles, in dark-colored, sealed Licaps,
with oxygen absorbers, making them highly resistant to both oxygen
(which, again, I really don't think is the issue) and UV (which might
be, minorly -- or more so, if one were for some reason not to keep one's
pills in an opaque container).

(NB: Sinclair has just today (Mon Dec 29 19:54:43 2003) sent an email to
the Science of Aging Knowledge Environment mailing list which indicates
that, despite having previously had a "consulting agreement with this
company", he has "terminated" said agreement because they have "been
using [his] name to promote their product ... [He has] taken steps to
ensure that they do not use [his] name to promote products in the future."
http://mailman.aaas.org/mailman/private/sagemail/20031229.txt

Despite his irritation with being used as a marketing plug, I doubt that
there were any frank lies involved in either document; I can only assume
that his consultancy did in fact involve verifying the STAC activity of
the Longevinex product & perhaps designing it in the first place
("product was developed under the direction of Dr. Sinclair" per the
press release). If he is indeed consulting on these points, then it's
hard to see what exactly he would object to, unless he just didn't want
his name used, period -- but it's hard to believe that he'd expect to
run a STAC assay and not expect it to be quoted or attributed to him).

COUNTERARGUMENTS

1. SAFETY. This dose of t-r is well outside any dose to which humans are
ever exposed in the diet. There is no epidemiological or RCT
justification for such a dose. On a "first, do no harm" basis, one might
be inclined to avoid the stuff.

This argument should IMO be taken seriously. There is absolutely nothing
stupider than to be a healthy person practicing the only
scientifically-justified life extension intervention who then shortens
hir life with a make-the-case dietary supplement. I am reasonably
confident of the stuff's safety in the sheer toxicological sense -- not
only becaue of the safety of the close cousin, quercetin, but because of
animal studies like (28-30). However, none of these really address the
issue of very long-term, moderately-high consumption -- the human case.

2. EFFICACY. WE DO NOT HAVE A 45 MO-OLD MOUSE! Yes, there are plenty
of mice being fed ravaging carcinogens and not developing cancer
following coadministration of t-r, but frankly this is irrelevant to the
case of long-term, chronic, low-dose use by humans living in a
reasonably clean environment.

What we do have is yeast, worm, and fly evidence. This sounds
interesting, but we must remember that yeast "aging" is not at all the
same thing as mammalian, & while worms & flies are much more interesting
models, still the upregulation of endogenous antioxidants,
administration of exogenous AOs, and hormesis also extend LS in these
critters, while these strategies have yet to pan out in mammals (though
they may square the curve).

According to the NYT and _Popular Mechanics_, there are rodent studies
under initiation. But of course, the simple fact that a study is
underway doesn't mean that it actually WORKS -- that's why you do a study!

This might mean that t-r supplements are a waste of one's $. Combine it
with safety concerns, and one might just want to not take the risk. (NB
that MOST supplements, beyond known essential nutrients at 1-2 times
RDA, share the same combination of this & the previous concerns. To
avoid anything beyond a very carefully-designed multivitamin/
multi-mineral (and NB that I've yet to see one that didn't have serious
flaws -- with the exception, to my astonishment, of Andrew Weil's New
Daily Multivitamin and Daily Antioxidant (!), which I recommend until
OrthoCore finally comes out from you-know-who [KH: AOR.ca]).

The best evidence to date that t-r has any bennies AT ALL is the "French
Paradox." Until recently, the "French Paradox" was really a bit of a
factoid: it's an ecologic/cross-cultural observation, not a proper
prospective finding, & it's not clear that it isn't just a time-lag
effect (31a). Plus, a 1996 meta-analysis (32) found that, from a cardio
health POV, "a drink is a drink."

HOWEVER:

(a) this was criticized (32a) because "Rimm and colleagues included in
their review 10 prospective population studies, of which three did not
analyze the effect of all three types of alcoholic beverage because
consumption of one or two of them was negligible. In two other studies
the analysis did not take into account the intake of the other types of
beverage. Of the remaining five studies, four suggested a greater
beneficial effect of wine ... In Rimm et al's study ... A breakdown of
the figures showed that the number of subjects who drank each type of
beverage were small. The exact data are not given, but we are told that
spirits were the beverage most frequently consumed, and the broad
confidence intervals give a hint of the small number of subjects in the
groups that drank beer and wine."; & more importantly,

(b) recently, some pretty solid, prospective epidemiology (33-38) has
been indicating that there may really be something to WINE consumption
per se: other boozes lower cardio, but not TOTAL, mortality, whereas
wine consumpption apparently does reduce death from other (&, when
assessed, ALL) causes.

Still, this could be just an antioxidant or detox enzyme-induction
mechanism; it hardly points to a CR mimetic per se. & also, there are
lifestyle & socioeconomic confounders to wine consumption, though most
studies are now controlling for this.

In any case, modest reductions in CVD & cancer from wine consumption (a)
don't PROVE that the t-r is what's responsible, & (b) do not demonstrate
a CR-mimetic effect!! The dose is too low, per analysis above, to
observe such an effect anyway. At least, however, it suggests that
SOMETHING in wine may be of benefit independent of alcohol content --
making wine (& PERHAPS t-r per se) of potential health benefit to CR
folk (alcohol per se in all probability mostly works via lipids &
fibrinolysis, & CRON folk tend to have excellent profiles on both parameters).

3. EFFECTIVE DOSING REGIMEN. Note that the dose & timing regimen above
is a series of extrapolations, & only covers PLASMA levels. Even if
correct, what about tissue disposition. A rodent study (31) in which
"[trans-]Resveratrol concentrations were measured in plasma, heart,
liver and kidneys" "after oral administration of red wine" found that
trans-resveratrol has "a significant cardiac bioavailability and strong
affinity for liver and kidneys." This should mean that concentrations in
the latter organs are disproportionately high, suggesting in turn a LOW
level of the compound elsewhere. Hard to say what the ramifications are
for LE effect, especially depending on time-course of selective,
tissue-specific accumulation.

4. CANCER. This is by far the most speculative -- and, irritatingly, the
most disquieting -- possibility. While it's thought likely that sirtuins
deacetylate a variety of proteins & hence modulate their activity, the
one that is best-characterized at present is p53, the cellular
proliferation protein. Indeed, it's deacetylation & deactivation of p53
that was used to test the effects of trans-resveratrol in human cells in
this study (1), and this & several other studies (39-41) show that
increasing SIRT1 levels or activity negatively regulates p53. & this is
where things may get a little creepy.

Cellular senescence is widely accepted to be an adaptation that exists
as an evolutionary adaptation against cancer. By shutting down cell
proliferation, the organism can prevent cells that are older or under
stress from going on to proliferate out of control & ultimately go
malignant. This is exactly what p53 does: it senses DNA damage or a
variety of stressors, and either moves the cell into senescence or
activates apoptosis.

As would be expected, then, increasing or activating SIRT1 under
conditions of stress prevents cellular senescence & apoptosis induced by
DNA damage and various stressors (1, 39-41). So the question is: might
preventing the activation of p53 cause a damaged cell, which would
otherwise have either shut down into senescence or committed cellular
suicide, buy it enough time not to be repaired (as Sinclair et al
propose, & as is the purpose of having this biological regulatory agent
in your system in the first place), but to go on to become a cancer?

Sinclair has considered this. He told the pop press (I'm doing this from
memory, now -- I can't find the article) that he isn't worried about
this, because "calorie restricted animals get less cancer" & thus
resveratrol, as a CR mimetic, would be expected to do the same; he also
noted the studies showing that trans-resveratrol is anti-carcinogenic.

The problem with the first half of this argument is that, of course,
it's circular: it remains to be demonstrated that sirtuins are involved
in the MAMMALIAN CR phenomenon, or more broadly that tans-resveratrol
is, in fact, a CR mimetic. One cannot invoke the mammalian CR phenomenon
to support the safety of a PUTATIVE CR mechanism and mimetic.

And re: the second argument: as with so many other supplements, the
mammal studies are not on SPONTANEOUS cancers, but based on a protocol
of dumping some hideous carcinogen into the animal's system & then
giving them the agent. Voila! Less cancer. But it's entirely reasonable
to assert that this is likely due to a simple antioxidant effect,
thereby protecting cells from having their DNA run thru' the Cuisinart;
this is quite distinct from any sirtuin-activating, p53-deactivating
mechanism, which happens (by definition) in cells which ARE subject to
stress &/or which have ALREADY had their DNA damaged. These kinds of
studies probably tell us very little.

The counterarguments: (1) several cell studies (as e.g. (18,42)) suggest
(but by their nature can't prove) that trans-resveratrol has anti-cancer
mechanisms quite distinct from antioxidant effects; (2) the studies
showing that wine consumers have less total mortality than other alcohol
drinkers (33,36-8) certainly suggest -- & (36-8) specifically found --
that SOMETHING in wine (& in particular the red stuff) has
anticarcinogenic activity; (3) there ARE studies (eg. (43-45)) in which
t-r has been shown to slow the growth &/or metastasis of IMPLANTED
cancers, while others (46,47) show no effect; these still don't address
the crucial window of crisis and transformation, in which the
hypothetical risk of down-regulated p53 would rear its head, but
(combined with the first 2 points) they do suggest that the OVERALL
effect on cancer risk is favorable or neutral.

MY JUDGEMENT: On the one hand, there is a serious case to be made to
just not take this stuff. Let's say it again: CR remains the only
intervention which doesn't involve gene therapy which has been confirmed
by repeated, independent studies to retard biological aging and extend
maximum lifespan in mammals. It seems that there is certainly something
foolish about gambling on a FURTHER extension of LS, when the
intervention is unproven & might, plausibly, SHORTEN your life for some
reason. This must be taken seriously.

That said...

I don't take any of the risks above (aside from the non-efficacy risk)
very seriously, for reasons already given. And I GREATLY value the
potential payoff. Adult-onset human CR is pretty damned weak medicine,
under any assumption less optimistic than that it will work BETTER in H.
sapiens than in Mus musculus (& if anything, the reverse seems likely).

I want those extra years; I CAN'T eat less, or I will seriously fear
damaging myself; the stuff LOOKS safe, and promising.

[KH: again the bottom line for an efficacious trans-resveratrol product is on that is] chirally pure, high-dose, [and] stability-protected.



1. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG,
Zipkin RE, Chung P, Kisielewski A, Zhang LL, Scherer B, Sinclair DA.
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a rat model.
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30. http://ntp-server.niehs.nih.gov/htdocs/Chem_Background/ExSumPDF/resveratrol.pdf

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38 Gronbaek M, Deis A, Sorensen TI, Becker U, Schnohr P, Jensen G.
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39. Langley E, Pearson M, Faretta M, Bauer UM, Frye RA, Minucci S,
Pelicci PG,
Kouzarides T.
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40: Vaziri H, Dessain SK, Ng Eaton E, Imai SI, Frye RA, Pandita TK,
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41: Luo J, Nikolaev AY, Imai S, Chen D, Su F, Shiloh A, Guarente L, Gu
W.
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44. 1: Miura D, Miura Y, Yagasaki K.
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[A1CR]

UPDATE (2005-02-03): MR now believes that the whole resveratrol-as-SIRT1-activator thing is a laboratory artifact:

Wake up! This was NEWS!

OK, no one [on the CR Society mailing List] has said anything about the post by [a regular poster] that started this
thread, or the central study largely reproduced therein (1). I can only
assume that some of you said, “Another damned full-text article by [this CRONie] …
probably off-topic … <DELETE>; that some of you started reading, found
it too technical, began to glaze over, and hit <DELETE>; and that a few
of you are sitting in stunned silence.

Folks, this study, unless massively flawed or fraudulent, says that the
whole rsveratrol-as-sirtuin-activator-and-thus-CR-mimetic thing is a
result of laboratory artifacts and that old nemesis of lifespan studies,
the short-lived strain!

Let me see if I can trim this down a bit. Ad hominem caveat: as The CRONie
noted, “5 of the 12 authors worked for Elixir Pharmaceuticals” –
Guarente’s commercialization wing, and the key rivals to Sinclair’s
Sirtris (& their collaborators, Biomol) in the time-wasting race to
develop sirtuin-based CR-mimetic life extension drugs (although as we’ve
seen, both have in fact retreated from such a goal to please the myopia
of venture capital). Sinclair’s group are the folks who reported the
sirtuin-activating and (apparent) life-extending effects of resveratrol
in yeast (2) and supposed life extension in fruit flies and C. elegans
roundworms, too (3).

-----------------
Resveratrol, a small molecule found in red wine, is reported to slow
aging in simple eukaryotes [yeast, worms, flies] and has been suggested
as a potential calorie restriction mimetic. Resveratrol has also been
reported to act as a Sirtuin activator [genes implicated in CR in yeast
and to a lesser extent rodnts and even humans] , and this property has been
proposed to account for its anti-aging effects.

We show here that resveratrol is a substrate-specific activator of yeast
Sir2 and human SirT1. In particular, we observe that, in vitro,
resveratrol enhances binding and deacetylation of peptide substrates
that contain Fluor de Lys, a non-physiological fluorescent moiety, but
has no effect on binding and deacetylation of acetylated peptides
lacking the fluorophore.


OK, you’re glazing . Fluor de Lys, described in perhaps too much
detail for most by Biomol’s Konrad Howitz at CR [Society] Conf III, is in essence
a molecular ‘tag’ designed to let researchers monitor via proxy what is
thought to be the key molecular action of sirtuins: the removal of
acetyl groups – which are gene-silencing when tagged onto DNA, and
activity-modulating when attached to proteins. Resveratrol’s claimed
ability to enhance sirtuin activity rests on its ability to stimulate
deacetylatioin of proteins tagged with FdL. What these folks found was
that the presence of the FdL tag ITSELF is central to the ability of
sirtuins to deacetylate these proteins: when, as in the real world, FdL
is not tagged onto peptides, then whatever structure-function alteration
which resveratrol imposes on sirtuins n longer results in enhanced
deacetylation. Ie, resveratrol does not, in fact, have the key molecular
action PRESUMED to underlie its life-extending effects in lower
eukaryotes, & indeed on whose basis Sinclair’s group started working
with it in the first place!!

Returning to the paper:

--------------
Consistent with these biochemical data, we find that, in three different
yeast strain backgrounds, resveratrol has no detectable effect on Sir2
activity in vivo, as measured by rDNA recombination, transcriptional
silencing near telomeres, and life span.
------------

This is a bit more readily understood. Some of the functions of sirtuins
which are thought or known to be connected to sirtuin-induced lifespan
extension in yeast just do not happen in vivo in response to
resveratrol. In fact, this was already observed by Sinclair’s group (2)
but didn’t raise enough of a flag. I mean, a lifespan extension is a
lifespan extension, right?

Yeah – unless there’s no LS extension! In Kaeberlein et al’s hands, “in
three different yeast strain backgrounds, resveratrol has no detectable
effect on … life span”!

Now what the heck is up with that? Mistake number 2: short-lived strains!

-------------

Sir2-independent life span extension by CR has been observed in the
long-lived BY4742 strain background; however, the majority of reports
examining CR in yeast have used the shorter-lived PSY316 strain
background. CR by growth on low glucose, or by several genetic models,
reproducibly increases life span in PSY316 by approximately 35%, while
growth in the presence of 10-100 µM resveratrol is reported to enhance
life span by up to 100% in this strain background ([2]).

We had found that, unlike the case for the BY4742 or W303R strains,
overexpression of SIR2 fails to increase life span in PSY316AR [ie, a
substrain of PSY316, in which resveratrol was reported to dramatically
extend LS -- by more, in fact, than CR apparently does, on a RELATIVE
scale (because it's a SHORT-LIVED STRAIN to begin with. So IOW, the
strain in which resveratrol dramatically increases LS does NOT enjoy a
LS advantagge from increased levels of Sir2 itself -- throwing, if
nothing else, a monkey wrench into the CR-mimetic-sirtuin-activation
hypothesis].

One possible explanation for this apparent discrepancy is that Sir2
[enzyme] activity is not increased in response to elevated SIR2 [gene]
dosage in PSY316. As previously seen for PSY316AR [ie, one substrain of
PSY316], overexpression of SIR2 had no effect on life span in PSY316AT
[another such substrain]. Sir2-dependent silencing of [two known sirtuin
target] genes integrated near telomeres WAS increased, however,
indicating that Sir2 activity WAS elevated in these cells. Therefore,
increased Sir2 activity, due to overexpression of the protein, failed to
increase life span in the PSY316 genetic background [again providing
strong evideence against the the CR-mimetic-sirtuin-activation hypothesis].

Since resveratrol is reported to increase life span in PSY316AT, but
activation of Sir2 does not increase life span in this strain, we
speculated that resveratrol might be acting as a CR-mimetic and
enhancing yeast longevity by a Sir2-independent mechanism. We therefore
tested the effect of resveratrol on life span in BY4742, a strain
background in which the longevity effects of CR and Sir2 are separable.
At a final concentration of either 10 µM or 100 µM [previously reported
to activate sirtuins & extend LS in teh PSY316 strain], *resveratrol*
obtained from Biomol Inc [gotta get that dig in there ] *failed to
significantly increase either mean or maximum life span*. Similarly, no
effect on life span was observed using a second source of resveratrol
obtained from Sigma.

[They then documented the lack of sirtuin-activating effect absent the
artificial Fluor de Lys tag].

Resveratrol was previously reported to increase life span by up to 100%
in the short-lived PSY316AT strain [2]. The inability of resveratrol to
increase life span in BY4742 suggested that resveratrol might act in a
strain-specific manner. In order to determine the generality of
resveratrol as a putative CR mimetic, we tested the effects of
resveratrol on life span in W303R, another short-lived strain commonly
used in yeast aging research. Unlike in PSY316, overexpression of SIR2
is known to increase life span in W303R (4).

However, similar to our results with BY4742, we were unable to detect
any significant increase in either mean or maximum life span of W303R
mother cells in response to resveratrol. In contrast to the prior report
[by Sinclair's group (2)], we also found that resveratrol had no
significant effect on rDNA recombination in W303R.

Given that we found no longevity effect from resveratrol treatment in
either BY4742 or W303R, we wished to reproduce the previously observed
[by Sinclair's group (2)] life span extension by resveratrol in PSY316.
We observed a *marginal* increase in life span in response to
resveratrol ...; however, the magnitude of the effect was much reduced
compared to that seen [by Sinclair's group (2)] (*12%* increase in mean
[replicative LS] [which is a dubiously-extrapolable LS measure IAC --
and see now (4), on which I've been planning to post for some time],
*versus 60-100% reported* ). Similarly, we observed only a slight effect
on life span in the PSY316AT strain (7% increase, p=0.29). Consistent
with the prior report (and unlike the case for overexpression of SIR2),
we also found that resveratrol has no effect on Sir2- dependent
transcriptional silencing in this strain.

-----------

This, of course, sounds more like a flat-out contradiction. However, as
I mentioned in my presentation at Conf III, the life-extending effects
of resveratrol in yeast reported in (2) was surprisingly variable, & the
effect on worms & yeast both widely variable & mostly quite piddley (3).
Without having directly compared the results, I'm not sure, but it may
just be that Sinclair's yeasts were kept even shorter than is natural
for this short-lived strain, further exaggerating the correction when LS
is NORMALIZED (not extended) by resveratrol.

We've seen that in mice a zillion times with antioxidants: BHT,
melatonin, etc etc. And indeed,

----------------

there is no direct evidence suggesting that resveratrol can activate
Sirtuins in vivo in either C. elegans or D. melanogaster. There are,
however, several examples of other antioxidant compounds reported to
increase longevity in invertebrates (e.g. (54-61)), suggesting that [the
modest] life span extension by resveratrol could result from its
antioxidant properties rather than its putative Sirtuin-activating
properties.

In addition to its antioxidant properties, resveratrol has also been
reported to specifically inhibit ... mitochondrial respiratory capacity
at complex III through competition with coenzyme Q. These activities of
resveratrol may be particularly relevant to its longevity promoting
effects in C. elegans, as several mutants with decreased mitochondrial
function have been reported to increase life span in this organism, and
decreased coenzyme Q levels, accomplished either through dietary or
genetic manipulations, have a similar effect. [This latter effect is
because, absent CoQ, these animals can't compensate by producing more
endogenously; CoQ is the site where most mtROS are generated, as it
"fumbles" electrons passed to it from Complex I, so reducing levels or
blocking it reduces mtROS and extnds LS in these critters]...

We suggest that further studies of the longevity promoting properties of
resveratrol should consider the full spectrum of biological processes
likely to be altered by this important compound. This will allow for a
mechanistic understanding of the effects of resveratrol on the life span
of invertebrates, and perhaps, mammals.

-------

After all of this, that is an awfully big "perhaps."

-MR


1. Kaeberlein M, McDonagh T, Heltweg B, Hixon J, Westman EA, Caldwell
S, Napper A, Curtis R, Distefano PS, Fields S, Bedalov A, Kennedy BK.
Substrate specific activation fo sirtuins by resveratrol. J Biol Chem.
2005 Jan 31; [Epub ahead of print] PMID: 15684413 [PubMed - as supplied
by publisher]

2: 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. Epub 2003 Aug 24.
PMID: 12939617 [PubMed - indexed for MEDLINE]

3: Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D.
Sirtuin activators mimic caloric restriction and delay ageing in
metazoans.
Nature. 2004 Aug 5;430(7000):686-9. Epub 2004 Jul 14. Erratum in:
Nature. 2004
Sep 2;431(7004):107.
PMID: 15254550 [PubMed - indexed for MEDLINE]

4. Minois N, Frajnt M, Wilson C, Vaupel JW.
Advances in measuring lifespan in the yeast Saccharomyces cerevisiae.
Proc Natl Acad Sci U S A. 2005 Jan 11;102(2):402-6. Epub 2004 Dec 29.
PMID: 15625107 [PubMed - in process]

[A1CR]

This is another important update to this thread (posted on behalf of MR; 2005-03-17):

Resveratrol Activation of Sirtuins an Artifact: Independent Confirmation

A new study (2) now confirms & expands the results of a previous study
by Kaeberlein's (apparently) independent group (1)...

... that the activation of sirtuins by resveratrol is a laboratory
artifact, resulting from the fact that the molecular "tag" ("Fluor de
Lys") that Sinclair's group has been using to detect the activation
(deacetylation) of various enzymatic targets by sirtuins actually
decreases the binding affinity of sirtuins to their target protein.

Resveratrol makes it easier for sirtuins to overcome the resistance
posed by the "tag"-bound protein. But take away the "tag," and there is
no enhancement of the activity of the sirtuin.

To use an analogy: imagine if you bought a contaminated supply of
gasoline, containing some evil chemical that gunks up your carburetor,
making your car run inefficiently. Now imagine that someone, looking to
design an efficiency-boosting fuel additive, just starts randomly
throwing chemicals into your gas line to see what might work. S/he hits
upon a substance that breaks down the noxious gak that's causing the
problem in your carburetor, causing your car to run normally again.

This normalization is made to look as if it were an actual /improvement/
in the normal functioning of your car, and is hailed as a boon to engine
longevity.

But as soon as you revert to pure fuel, you find that the miracle
additive is of no benefit, because the hindrance that it removed isn't
there in the first place.


Bottom line: whatever the place of sirtuins in the CR effect in mammals
(still an open question, though there remain promising hints), resveratrol
is not a shortcut to their activation in real-world conditions.

-MR


1. Kaeberlein M, McDonagh T, Heltweg B, Hixon J, Westman EA, Caldwell S,
Napper A, Curtis R, Distefano PS, Fields S, Bedalov A, Kennedy BK.
Substrate specific activation fo sirtuins by resveratrol. J Biol Chem.
2005 Jan 31; [Epub ahead of print] PMID: 15684413 [PubMed - as supplied
by publisher]

2: Borra MT, Smith BC, Denu JM.
Mechanism of human SIRT1 activation by resveratrol.
J Biol Chem. 2005 Mar 4; [Epub ahead of print]
PMID: 15749705 [PubMed - as supplied by publisher]v