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Reversing Aging
Rapidly
with Short-Term Calorie Restriction
Continued
from...
L.E.: Here's a difficult question. We know that long-term
calorie restriction extends life span, but since short-term
calorie restriction only reproduced some of the changes that
long-term calorie restriction produced, how can you predict
the effect of short-term restriction on life span?
S.S.: My assumption is that the effects of calorie restriction
are linear. By this I mean that if you are able to improve
the expression of 70% or 60% or 50% of the genes and return
them to the level of expression that life-long calorie restriction
produces, then my assumption is that you're going to get 70
or 60 or 50% of the effects of long-term calorie restriction.
My reason for thinking this is that it seems that what matters
is general gene patterns rather than specific genes. Between
different tissues, you don't find that the exact same genes
change, but you find that genes that are in the same kinds
of pathways and that are involved in the same kinds of responses
change. And so my guess is that calorie restriction creates
beneficial patterns of gene expression. At this point, though,
it's just an opinion. Ultimately, we'll have to do more studies
and follow up to find out whether or not these short-term
changes really do also delay the onset of age-related diseases
and extend life span.
L.E.: This starts to get us into the area of how you can
apply your findings clinically and what the implications are
for clinical intervention. Short-term calorie restriction
is obviously more attractive to most people than long-term
calorie restriction, but we've also heard that yo-yo dieting,
in which you repeatedly get rid of a lot of weight for a while
and then gain it back again, might be harmful.
S.S.: My current understanding is that the studies say that
it's not harmful to do that. My personal opinion is that you
should do whatever you need to do to get the weight off, short
of something that would hurt your health. I don't think that
you should try bulimia or drugs that could harm you, but I
think that people would be well advised to do whatever they
can to get the weight off.
L.E.: That's very encouraging. I'm sure you've just made
a lot of people happy.
S.S.: A recently published study indicates that if people
will lose ten pounds, regardless of what their weight is before
they start the diet, then many of their physiological parameters
of health will improve. It improves your glucose sensitivity,
lowers your blood glucose, lowers your blood insulin levels,
improves your heart rate, improves your blood pressure. So,
even losing weight for a short period of time has beneficial
effects.
L.E.: Okay, so is it theoretically possible to use short-term
calorie restriction to partially reverse aging in very old
humans?
S.S.: Well, I can't tell you for sure, but my guess is yes.
Not necessarily to reverse aging, but to improve health and
physiology and to slow aging.
L.E.: Based on your mouse study, wouldn't that almost necessarily
involve a partial reversal of age-related changes?
S.S.: Yes, it would. I hadn't thought about that, but I suppose
that's true.
L.E.: Of course, there might be hazards associated with asking
95 year-olds to eat less. They may have trouble staying alive
as it is. Some people who are really old need more calories.
S.S.: I've had people ask me about using caloric restriction
for cancer patients or for very elderly people, and my advice
is always not to try it. Calorie restriction is something
that's very well characterized in animals and rather poorly
characterized in humans. We are not animals in a vivarium.
We have to go out and cope with a very complex world, and
we have to have energy and strength to do it. There's no question
that under-eating improves our health, but I don't think that
you should take sick people and try to improve their health
by under-feeding them.
L.E.: Including a person who might be considered sick precisely
because he or she is extremely old and debilitated?
S.S.: Yes.
L.E.: So there may be limits beyond which you can't go?
S.S.: There may be.
L.E.: This suggests the need for a more practical alternative.
Since almost nobody wants to be on calorie restriction anyway,
and since it does have its safety issues and inconveniences,
there is a desire on the part of many people to develop what
are called calorie restriction mimetics, in other words, drugs
that imitate the effects of calorie restriction. You refer
to that in the paper, and the fact that your results provide
an opportunity for screening drugs and for finding the magic
pill that would simulate calorie restriction.
S.S.: To the best of my knowledge, gene chip analysis would
be the fastest way of doing a first screen for drugs and treatments
that mimic the effects of caloric restriction.
L.E.: Are you in fact screening any potential candidates
at this time?
Staff scientists Patricia
Mote (left) and Joseph Dhahbi, Ph.D. (right) watch as
Dr. Spindler removes frozen tissue specimens from liquid
nitrogen storage.
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S.S.: Yes, and we're preparing to screen others, too. We're
also preparing to screen compounds for their effects on gene
expression for other companies. I think the gene expression
assay for the first time provides a method for demonstrating
the extent to which a given agent is capable of reproducing
the effects of caloric restriction on these genetic expression
biomarkers of aging and thereby predicting the agent's ability
to reproduce the beneficial physiological effects of calorie
restriction. So, I think it's very exciting. Subsequent studies
can be done to verify that compounds that pass this biomarker
screening are in fact effective in preventing the onset of
age-related diseases and extending life span. Excitingly,
gene chip biomarker studies can be done in humans after the
preliminary studies are done in animals. The screening of
humans for delaying the onset of age related diseases with
these compounds is a very real possibility.
L.E.: So if you find a compound that's effective in a mouse
or in a monkey or whatever, you can find out right away if
it has the same gene expression effects in a human.
S.S.: Yes, you can.
L.E.: So not only do you have a technique here that can give
you a comprehensive look at virtually the whole genome, but
what's even more revolutionary is that it can do so for aging
intervention tests in a short time.
S.S.: That's really the contribution of our technology-it
makes the initial screening rapid.
L.E.: This has really been the bottleneck that has held up
the entire field of interventive gerontology: it's just not
practical to test somebody for their whole life to see if
they live longer or not. Now you have a solution to that problem.
S.S.: Even to test an animal for an effect directly on life
span takes more than three years in a mouse. Others have produced
screens that they say would take a year or ten months. But
if you can screen in four weeks, the number of screenings
that you can do increases enormously, and the cost goes down
dramatically.
L.E.: Talk a little about the company you are working for
that is developing the commercial applications of your work.
S.S.: We founded a company called LifeSpan Genetics, which
is currently funded by the Life Extension Foundation. LifeSpan
Genetics has licensed the commercial rights to the three seminal
patents that have been applied for in the field, two from
the University of Wisconsin, one from the University of California.
We are testing drugs for their calorie restriction mimetic
effects and are continuing to look at the effects of different
periods of caloric restriction in many tissues in both mice
and monkeys, and we are also planning studies in humans.
L.E.: It used to be that the Life Extension Foundation would
quietly support research in labs that produced no fanfare,
or would report research from other labs that was not funded
by LEF. So it's very gratifying that now we're able to report
on research that was funded by the Foundation and that has
not only made it into the prestigious Proceedings of the National
Academy of Sciences, but has also been singled out for special
attention by that journal. I understand that stories on your
work are appearing on CNN and Science Now as well as in the
Wall Street Journal, The Washington Post, the journal Nature,
the Reuters News service, and other media sources. Congratulations!
It's very exciting that the Foundation is now helping to advance
what are probably the most powerful anti-aging tools ever
available!
S.S.: For many years the Life Extension Foundation has funded
studies of ours and of others that were published in scientific
journals. But now through LifeSpan Genetics (www.lifespangenetics.com)
we have two papers, not only the PNAS paper but also one soon
to be published in the Journal of Nutrition, which represent
work that is patent-pending and licensed by the company for
work in identifying anti-aging CR mimetic compounds and treatments.
L.E.: From a practical point of view, how would you approach
clinical application of your observations? I assume it's hard
to take liver biopsies from humans, so what would you do?
S.S.: There are tissues in humans that you can assay readily.
And I think there's a tremendous amount of information that
can be gleaned from gene chip assays of those tissues, especially
since studies are being done in both monkeys and mice. We
perhaps won't have to be as thorough in humans once we have
this background of knowledge from our non-human primate relatives.
L.E.: What are the prospects for using gene expression profiling
to develop a biological measurement of aging? In other words,
a way of giving an individual a reasonable assessment of what
his or her biological age is.
S.S.: Gerontologists have been interested in developing a
biomarker system for measuring biological age for at least
twenty years, that I know of. And they want to do that for
precisely the reasons we've talked about in this interview.
One would like to be able to know when a treatment or compound
is affecting aging without having to look at life span. Also,
just from every human's point of view, we'd like to know how
close we are to death. I don't know how well gene expression
profiling will serve as a yardstick for biological age. It's
a little early to tell yet, but I think because we're looking
at so many things, gene expression levels eventually of 30,000
genes and 3.1 splice variants each, some of which will change
with age, I can't help but think it's likely we'll find highly
reliable biomarkers of aging.
L.E.: Agreed. The magnitude of the problem of dealing with
aging may depend on how many genes are involved. Can you speculate
about how many genes you think may change with aging in a
whole mouse or a human? Based in part on the numbers seen
so far in your lab, and based on the fact that there is overlap
between the genes that changed with age in your study and
the genes that change according to results in other labs,
it seems the total number of changes could be very low.
S.S.: It is an amazingly small number. Most gerontologists
agree that there are on the order of tens to hundreds of genes
that are involved in changing the life span of an organism
from shorter to longer, based on selection experiments (directed
breeding to produce long-lived animals). It has to be a relatively
small number of genes because you're able to select for long
life over a relatively short period of time.
L.E.: In the initial Weindruch and Prolla paper on muscle,
the number of genes whose expression went up with age by more
than a factor of two was 58 out of the 6347 genes examined,
or 0.9 percent. The number of genes whose expression went
down by more than a factor of two was 55, or also about 0.9
percent. The total percentage of genes that went either up
or down by over 2-fold was 1.8 percent.
S.S.: You have to remember that that was 1.8 percent of the
total probes that were on the chip. I don't know about those
studies in particular, but normally in gene expression studies
done with micro-arrays, the number of genes that actually
report a signal (are active) is significantly less than the
total number of genes on the chip. I don't know what their
results were in terms of active genes, but I think the number
of genes that change with age is probably a larger percent,
several percent, of the genes that are active.
L.E.: OK. How many of the genes were active in your study?
S.S.: Oh, around 4,000, or about one third of the total looked
at.
L.E.: OK, now let's try to compare the muscle results with
your liver results. First, you examined over 11,000 genes
and found only 46 known genes that went either up or down
with age.
S.S.: Yes. I was surprised to find, looking at the liver,
that in control mice there were only 20 that went up and 26
that went down. And there were far fewer changes when we imposed
calorie restriction.
L.E.: It's astonishing, isn't it?
S.S.: It was astonishing to me.
L.E.: On a percentage basis, those 46 genes represent 0.9
percent of the known genes on the chip, around 1.1 percent
of the active genes on the chip, and just 0.4 percent of the
total genes on the chip. These age-related changes in liver
appear to be considerably fewer than what was found in muscle.
At the rate of 1.8 percent of the genes on the chip, one would
expect a total of 198 total genes, or perhaps 109 known genes,
to change in expression with age in the liver. However, you
saw only 46, which is less than half of this predicted number.
And yet you considered a change as small as a 1.7-fold change
to be meaningful, whereas Weindruch and Prolla used a more
restrictive detection threshold of over 2 fold (though they
also reported some results with thresholds as low as 1.5-fold).
Therefore, if anything, you would have found even more than
109 changes in known genes if aging had affected both tissues
to the same extent, but you did not.
S.S.: I thought that we would see more transcription factors.
I thought that we would see more fundamental regulators in
gene expression changing. Aging, at least in the liver and
in the muscle and in the brain, has a fairly subtle effect
on gene expression.
L.E.: On the surface, everything seems to change with age,
but at a deeper level, relatively few things change. As you
said, aging is subtle.
S.S.: On the gene expression level it looks subtle. Now on
the protein level it may be different because there's a lot
of regulation that involves phosphorylation cascades and other
kinds of modifications of proteins, and we know very little
about that.
L.E.: On the other hand, calorie restriction may take care
of a lot of that. In your study, for example, only 3 out of
the normal 20 age-related increases in gene expression (15
percent) escaped correction by either long- or short-term
calorie restriction! Two gene expression increases were reduced
about 50 percent, and 15 increases were totally abolished
by either long or short-term restriction. Of the 26 decreases
in gene expression, 13 (50 percent) were blunted by long-term
calorie restriction and 18 (69 percent) were blunted by short-term
calorie restriction, leaving only 5 decreases (19 percent)
unaffected by either long or short-term restriction. These
results are amazing.
S.S.: Yes. I agree.
L.E.: Going further, it seems that a large fraction of the
changes you saw with aging don't necessarily represent actual
liver aging per se. Instead, they largely appear to represent
changes that increase disease susceptibility. This means that
the liver actually showed really very few true aging changes.
S.S.: We did see changes that look like the changes that
you see in the development of age-related diseases. This has
been found by other workers conducting micro-array gene expression
studies in other tissues as well. I think our results are
very consistent with theirs in showing that gene expression
profiles in tissues begin to resemble profiles of tissues
that have age related disease processes going on in them.
Our tissues looked healthy-we could slice them and look at
them under the microscope and see no signs of liver fibrosis
for instance. But when we looked at gene expression in these
tissues with age, we found changes that more and more resemble
those that you see in diseased tissues. So, I think that's
part of the development of age-related diseases-a drift towards
gene expression that resembles the gene expression of diseased
tissues. Calorie restriction reverses much of that, short
and long-term.
L.E.: For example, a major fraction of the changes brought
about by calorie restriction in your study had the effect
of helping to prevent cancer from getting out of control.
S.S.: Most mice living under laboratory conditions die of
cancer.
L.E.: So it seems that another possible practical spin-off
of your work could be in the area of cancer prevention.
You point out in the paper several specific examples of how
changes produced by calorie restriction might block cancer.
In fact, those insights might allow the development of anti-cancer
drugs that, on the face of it, have nothing to do with calorie
restriction per se.
S.S.: I don't know if our agents will be anti-cancer drugs
in the conventional sense of drugs for treating pre-existing
cancers, but what I can see clearly is that we're going to
be able to develop preventatives. Not just for cancer. Of
course, it would be a major step forward if we developed prophylactics
for the development of cancer. But we can also attack other
age-related diseases, because caloric restriction delays the
onset and reduces the incidence and the severity of many age-related
diseases. So I think we have the potential, in searching for
treatments that reproduce these biomarkers of aging, of finding
mimetics that will affect the rate of onset, the severity,
and the incidence of many diseases.
L.E.: The beauty of CR is that it has such a broad effect
against aging, hitting so many different pro-aging systems.
But after you figure out what each specific CR gene effect
is, you may find that you want to tailor a variety of drugs
that are specifically targeted at those individual effects,
rather than trying to replicate all CR effects at once.
S.S.: That's right. We don't really know yet if we'll find
mimetics that will reproduce all of the effects of CR in all
tissues. I think it's probably more likely that we're going
to find treatments that reproduce some of the effects and
are tissue-specific in doing that. I think we'll probably
end up having to combine mimetics in order to achieve the
full effects of caloric restriction.
L.E.: In your paper, you only reported changes in genes whose
functions are known, except for maybe one case, which was
the major urinary proteins, which were not really discussed.
S.S.: We didn't report what are called ESTs (expressed sequence
tags).
L.E.: Did you see any EST changes?
S.S.: Oh, yes. But we just don't know what they mean. First
of all the function of the ESTs is not known. Very often it
is not known whether different ESTs even represent different
genes. Some certainly represent the same gene.
Some ESTs are constant regions of antibodies.
L.E.: This points up the disadvantage of not having the complete
Rosetta stone of biology - the complete genome-on a chip yet.
In other words, there may be a lot of genes that are even
more important than what's been observed, but they're just
not on the chips yet.
Dr. Dhahbi and Patricia
Mote prepare to measure gene expresson on an agarose
gel.
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S.S.: They're coming. It will only, I think, be a year or
maybe two before we have a whole genome set on chips that
will give you the gene expression levels of all of the genes
and all of the splice variants. There may be 30,000 genes
in the mouse and the human, but each one of those genes gives
rise to, on average, 3.1 different messenger RNAs (mRNAs),
the splice variants. So in many cases with the probes that
are available now, we don't even know which splice variants,
which of those three possibilities, we're assaying. But the
commercial chips are getting better and better, and we're
learning more and more about the genes as the Genome Project
is continuing to yield information.
L.E.: Will the complete genome and variants chips be available
for both humans and mice?
S.S.: For humans as well as mice.
L.E.: Fantastic.
S.S.: And then the field of proteomics (large scale protein
surveying) is growing and expanding, and the technology is
improving dramatically there. So I think the next ten years
are going to give us a rich picture of what's going on during
aging in different tissues and how that's affected by caloric
restriction.
Continued...
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