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Reversing Aging
Rapidly
with Short-Term Calorie Restriction
Life Extension Foundation-funded Research
Breakthrough Published in the Proceedings of the National
Academy of Sciences
An Interview with Stephen R. Spindler, Ph.D.
Stephen R. Spindler, Ph.D.
of the Dept. of Biochemistry at the University of California
at Riverside (UCR) directs the genechip studies conducted
by LifeSpan Genetics.
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On Tuesday, Sep. 4th, the Proceedings
of the National Academy of Sciences (PNAS) web site features
a paper from the laboratory of Dr. Stephen Spindler, who has
been probing the life-extending effects of calorie restriction
using advanced gene chip technology. (For an explanation of
gene chip studies of aging, please see our interview with
Drs. Tomas Prolla and Richard Weindruch in the November, 1999
issue of Life Extension Magazine.) Dr. Spindler examined aging
changes in the expression of 11,000 genes and the modification
of these changes by calorie restriction. The major conclusions
from this study are that many of the life extension effects
of calorie restriction happen rapidly, and that these effects
can be shown not only in young animals, but also in old animals
not previously on calorie restriction. Calorie restriction
not only slows aging and extends maximum life span, but it
partially reverses aging changes as well! On top of that,
the fact that calorie restriction acts rapidly means that,
for the first time, it is possible to test anti-aging interventions
in weeks rather than years, which should drastically accelerate
the search for anti-aging treatments. Dr. Spindler, who is
a professor at the Department of Biochemistry at the University
of California at Riverside and works for a company called
LifeSpan Genetics, was interviewed about his results by Dr.
Gregory M. Fahy and by Life Extension Foundation founder and
president Saul Kent on August 17th, 2001.
Life Extension: Dr. Spindler, what is the essence of your
new observations, which are just coming out in PNAS?
Stephen Spindler: I think the conclusion you can reach from
the paper is that even in very old animals, caloric restriction
will very rapidly produce most of the gene expression effects
that you see in long-term calorie-restricted animals. That
means, I think, that even in the short-term, older people
may be able to benefit rapidly from switching to a calorically-restricted
diet, and that fits with some of the information that has
been in the literature for years. For instance, type II diabetics
improve when they start under-eating. Their blood glucose
levels improve. Their insulin sensitivity improves. Their
general health improves, even before the fat mass, for instance,
is depleted. So, there have been some hints that underfeeding
could produce positive effects rather rapidly, but this research
that we are publishing shows this for the first time, directly,
using gene expression profiles as biomarkers for the effects
of caloric restriction.
L.E.: Are you the first to actually look at the biological
effects of calorie restriction using gene chips?
S.S.: No, the first studies were done by Drs. Richard Weindruch
and Tomas Prolla at the University of Wisconsin. Our interest
has been the rapid effects. We are interested in the transition
from one state to the other. Our contribution here has been
looking at how rapidly the effects of caloric restriction
are established in animals that have been allowed to eat almost
all that they wanted for their entire lives, like most people
do.
L.E.: Could you please describe how the short-term calorie
restriction experiment was actually done?
S.S.: We took a group of animals that had been allowed to
eat almost all they wanted their whole life and we intervened
when they were quite old - 34 months of age. These mice would
be the equivalent of people who are probably 70 or 80 years
old or older - I'm just guessing at the human equivalent age.
We took a group of them and said okay guys, the party's over,
it's time to diet. We under-fed them first for two weeks by
20% -- that is, 20% less than they had been eating previously-and
then for two weeks after that we fed them an additional 20%
less so that for the second two weeks they were eating 40%
less than they had been eating most of their lives. At the
end of that time, at 35 months of age, we sacrificed all of
the animals. We then compared the gene expression profiles
in the livers of these mice to those in four other groups
of mice. The old controls were mice that always ate almost
all they wanted until being analyzed at 27 months of age.
The long-term calorie restriction mice were those mice who
had spent their whole lives being under-fed by 40% until the
age of 27 months. Finally, the short-term calorie restricted
mice were, as I mentioned, switched from fully fed to under-fed
for just four weeks, and even at that only two weeks with
"full strength" calorie restriction. We also had a young (7
month-old) control group and a young long-term calorie restricted
group (also 7 months old) so we could look at calorie restriction
independently of aging.
L.E.: Since you started your short-term calorie restriction
experiment at 34 months and let the mice run out to 35 months
before you checked them, they were actually 8 months older
than your long-term calorie restricted animals, which you
checked at 27 months.
And on top of that you allowed only two weeks with full strength
calorie restriction. That hardly seems fair to the short-term
calorie restriction group, and yet you saw a lot of beneficial
changes anyway.
S.S.: One of the problems with doing experiments of this
kind is that it is very hard and expensive to get very old
groups of mice. So sometimes we have to make comparisons between
old mice that are of slightly different ages, but I think
the results are still valid. It's true, though, that because
the short-term restricted mice were 35 months old, we might
not have been able to appreciate fully all of the effects
of late, short-term calorie restriction.
L.E.: What fraction of animals would normally be alive at
34 to 35 months of age in your population?
S.S.: I would guess we're probably down to 35 or so percent
of the animals surviving at those ages. We've actually taken
two inbred lines and crossed them, so that we get a vigorous
mouse that has no genetic defects that cause it to have a
shortened life span. They are the longest-lived mouse strain
of which I'm aware.
L.E.: So if you see an anti-aging benefit in these mice,
it's a true anti-aging benefit, not just a correction of some
life-shortening genetic defect. That's as good as it gets
in studies like this. Now, let's attack this from a slightly
different angle. Since the animals were already extremely
old when you imposed short-term calorie restriction on them,
and since their gene expression profiles appeared more like
those of young animals after short-term calorie restriction,
it seems inescapable that calorie restriction is not only
able to slow age-related changes, but that it is able to reverse
age-related changes as well. And it is able to do so over
a remarkably short period of time.
S.S.: I think that may be our most significant contribution
here.
L.E.: Has anyone else ever suggested that calorie restriction
could reverse aging, not just slow it? Or is your finding
truly unique?
S.S.: As far as I know, there had been no suggestion in the
literature before our study that calorie restriction could
reverse age-related changes in gene expression. I think the
assumption has been that it prevents deleterious age-related
changes in gene expression. It had been our assumption as
well, and we've published a number of papers on gene expression
where we just assumed that calorie restriction was preventing
deleterious changes. What these studies showed for the first
time was that in fact that assumption was incorrect. Calorie
restriction can reverse the majority of the deleterious age-related
changes in gene expression that we found.
L.E.: That's revolutionary and very interesting.
S.S.: There's another issue here too, and that is that we
only did two weeks of extreme caloric restriction and two
weeks of mild. We're now looking to see if we can find early
responding genes, late responding genes and genes that may
be in the middle, or genes that may require life-long caloric
restriction in order to prevent a change.
L.E.: So, for example, if the short-term calorie restriction
were made a bit longer, it might work even better.
Dr. Spindler in the UCR
vivarium where the
research animals are cared for.
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S.S.: That's true. I think there's the chance too that if
we do this in younger animals, it is possible it will be even
more effective, since they will not have accumulated damage
throughout their lives beforehand.
L.E.: There would be less damage to reverse.
S.S.: Yes.
L.E.: In general, is it true that caloric restriction started
earlier in life, if done properly, leads to longer life span
extension and stronger anti-aging changes?
S.S.: There are papers in the literature that indicate that
it is true that calorie restriction earlier in life has a
bigger impact on lengthening life span and decreasing the
onset of age-related diseases than even longer calorie restriction
imposed later in life. Nevertheless, our study shows that
very late in life in very old animals calorie restriction
will rather quickly start to reverse bad changes in gene expression
and send them back to youthful levels of gene expression,
affecting genes that we can be pretty confident are going
to improve the physiology of the animal.
L.E.: So it's not too late for the older folks out there,
for people who think they're over the hill, to do something
about aging. There's still hope.
S.S.: That's the best news for me.
L.E.: For me, too. What is the oldest age at which caloric
restriction had been previously found to lead to an extension
of maximum life span?
S.S.: In mice, the oldest study I know of was started at
15 months of age. In fact that was part of the study being
supported by the Life Extension Foundation that was done by
Richard Weindruch and myself. We've started another study
at the University of California even later in life, and we'll
find out whether starting mice on calorie restriction much
later in life will have a lifespan-extending effect.
L.E.: There is a fascinating paradox in your paper. According
to some of the tables in your paper, starting calorie restriction
in old animals did not reproduce all of the benefits of long-term
calorie restriction. Nevertheless, some of the benefits of
short-term calorie restriction in the old animals were actually
stronger than with long-term calorie restriction! That's a
rather striking result. Short-term calorie restriction was
even more powerful than long-term calorie restriction in some
cases. Can you explain this?
S.S.: I think we'll understand better what those many early
effects mean when we determine whether they're maintained
after acute onset of calorie restriction or whether they're
only transient. I think we need to know more about later times.
We've looked at four weeks of calorie restriction, and we're
looking at other shorter and longer durations of calorie restriction.
L.E.: So the super-protective effects of short-term calorie
restriction may recede as you go out longer, returning to
being more like long-term calorie restriction. But short-term
calorie restriction actually had some aging reversal effects
that did not show up with long-term calorie restriction at
all. Four cell cycle genes that did not respond to long-term
restriction were totally corrected by short-term restriction,
and the same was true for 5 genes in your "others" group,
including the anti-atherosclerosis gene, apolipoprotein E.
Short-term restriction reversed increases in two stress genes
(HSP-25 and stress-induced phosphoprotein 1) that were not
touched by long-term restriction, but failed to correct two
gene expression increases in your "inflammatory response"
category that were completely reversed by long-term calorie
restriction (CR). It's remarkable that you actually saw a
reversal of many age-related changes with short-term CR that
were not prevented with long-term CR.
S.S.: Yes, that is true. And we saw that even with shorter
times of calorie restriction.
L.E.: Even shorter than you showed in the paper?
S.S.: Yes.
L.E.: Incredible. Is it possible that even though shorter
term calorie restriction doesn't produce the same total number
of anti-aging changes that long-term restriction does, it
nevertheless causes the most important such changes?
S.S.: We don't know yet, really, which are the most important
gene expression changes. But we do know that the changes produced
by short-term calorie restriction apply to the same categories
of genes that are affected by long-term restriction and affect
the majority of the same genes that are affected by long-term
CR. So I think it's a very high probability that short-term
reproduces the majority of the long-term effects.
L.E.: How reliable are the magnitudes of the changes you
reported? If you saw a 4-fold change in a given result with
short-term calorie restriction and a 2.5-fold change with
long-term calorie restriction, how likely is it that these
changes are really different and don't just differ because
of random statistical fluctuations?
S.S.: We found that the changes we detect with Affymetrix
gene chips (the ones used in the PNAS study) are really pretty
reliable. It is true that you can't do very good statistics
with these Affymetrix chip studies. The reason for this is
that the chips and chemistries for the chips are so costly.
Also, old animals are expensive and difficult to get. Because
our funding has been limited, we haven't been able to include
the numbers of animals in each group that we would like to
have to do traditional statistics. So we've been in a situation
where we're able to measure 1,000 to 10,000 times the number
of genes we used to be able to measure, but we don't routinely
do as many samples as we used to do. So traditional statistical
tests are difficult. The tact most users of this technology
have taken is that you validate a cross section of the results
that you get using another technology that's been proven to
be highly reliable. We've used a technique called Northern
blotting in order to validate our changes. What we found was
that at least 95% of the changes that we detected with the
Affymetrix chips were reproduced using Northern analysis.
So, my guess is that the results we found are reliable. Whether
a 2½ fold change is the same or less than a 4 fold change
is difficult to say, but the fact that both changes are in
the vicinity of two to four fold is highly likely to be correct.
L.E.: In the tables in your paper, if you report, for example,
a two-fold decrease in expression of a given gene due to aging
and a two-fold increase in expression of that same gene in
old animals after long-term calorie restriction, does that
mean that there was no change with age in the calorie restriction
group compared to a young control mouse?
S.S.: Though it may not have been clear in the way the data
were expressed, if it went down by two-fold with age and calorie
restriction increased it by two-fold, we have returned it
to near the youthful baseline level.
L.E.: The reason for the question is that in some cases,
you might see a two-fold drop with age and a four-fold increase
with calorie restriction!
S.S.: Again, I don't know if it really is four-fold greater
in that particular case. You have to look at these gene chip
studies as being screening studies. Probably two-fold and
four-fold changes are different, but until you test each change
with another technique, using large numbers of animals and
get statistics that are highly reliable, it's a little difficult
to say. What you can say is they both changed in opposite
directions to about the same fold and you can take that to
the bank.
L.E.: Okay.
S.S.: Any gene chip study is really an initial screening
event. It can show you that things that you might never have
expected to change do change, and it can point you in new
directions that you might never have known about, because
it's an unbiased screening of a large percentage of the total
genes that the organism expresses. When you have those changes,
then you can be pretty sure, if you're looking at changes
that are 1.7-fold or greater, as we did, that the changes
are real. The expression levels change up or change down and
they change in the approximate percentage that we found. But
that doesn't mean that more detailed studies aren't warranted.
Continued...
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