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Reversing Aging
Rapidly
with Short-Term Calorie Restriction
Continued
from...
L.E.: So the prospects for intervention appear to be good,
but now let's talk about some of the problems of interpreting
and using gene expression biomarkers as indicators of successful
intervention into aging. First, it's very possible that a
real calorie restriction mimetic will not push all of the
genetic buttons that calorie restriction itself pushes. It
may push only some of them. Given imperfect mimicking of calorie
restriction, do you have in mind any hierarchies of the relative
importance of different gene effects that you would use to
distinguish a better calorie restriction mimetic intervention
from a less good one?
S.S.: Yes. I think we have to look at each tissue and use
what we know about the physiology of the tissue with aging
and the effects of the change in gene expression that we see
and compare that to the effects after long-term calorie restriction.
In every tissue the effects are different, but there is a
vast literature about the physiology of the tissues with aging
and with caloric restriction. This can provide enormous help
in interpreting the results that we get.
L.E.: Have you compiled all of that literature into some
rules of thumb? This seems like a commercially attractive
possibility for a company, because it seems you could even
patent such rules of thumb if you discovered them. Can you
tell us whether you've developed such rules of thumb and if
not, if you're working on that?
S.S.: Certainly we're working on that. It's a complex process
and one that takes studies in a lot of tissues because every
tissue is different so far. There are even differences between
species that we've found, and that have been found by others.
So, it's going to take some work to establish clear rules.
But, yes, they're coming along. We have a good idea of what
the effects are in liver, and now we're looking beyond that
into other tissues as well.
L.E.: Your task would also be simpler if you could place
the changes you see within some kind of hierarchy of cause
and effect as well, so you would be able to say which age-related
changes are primary and which ones are secondary. Do you have
any feeling as to whether you can identify such a hierarchy
of changes, so you can tell whether some of the aging changes
you saw may depend on other gene changes, as opposed to being
primary changes?
S.S.: That is a difficult question. One of the ways we're
trying to get at the answer to that question is to look at
various times after the onset of caloric restriction: at the
changes that happen early, at a middle time, and late. Changes
that appear early and disappear, and changes that appear early
and are also present in the long-term state. I think that
these kinds of studies are going to give us a better feeling
for which may be primary changes and which may be secondary
changes. We can use bioinformatics to cluster the genes that
change and start to ask which regulators are likely to be
responsible for those changes. That will allow us then to
do fundamental experiments that will ask more directly, do
these specific genes affect health span and life span?
L.E.: That's very exciting. Another problem for interpretation
of gene profiles might arise from the fact that very similar
proteins can react in different ways to the same condition.
For example, heat shock protein number 70 (HSP-70) doesn't
seem to be affected by aging in your experiment, whereas HSP-86
and HSP-25 both go up with age.
S.S.: Proteins like these don't act alone, they act in groups.
For HSP-70, where we didn't see an effect with age, it may
be that there is a rather subtle effect. When we look at the
livers of starved and fed animals, we find that HSP-70 responds
strongly to feeding. After you eat, the level of messenger
RNA for HSP-70 goes up about three-fold in a spike (peak),
and then that decays after the meal. How many of those spikes
you have and how high they are and how often they occur will
affect the time-averaged level of HSP-70. If you fast an animal
for a long time, the level of chaperone proteins tends to
fall. So calorie restriction may have a subtle effect on some
chaperones that we haven't picked up with these studies, where
we looked at animals that had been fasted for 24 or 48 hours,
because we missed those spikes that determined the averaged
level of the protein over the long-term. So, these are very
subtle and complex effects at work.
L.E.: It's a point we don't usually think about, but a lot
of the things you might be measuring depend upon what time
of day it is, how long it's been since you last ate, that
sort of thing. The genome is very dynamic and if you look
at different times, you'll get different results.
S.S.: That's so true.
L.E.: At least you're developing the tools to deal with the
complexities.
S.S.: That's right.
L.E.: Another problem with using gene expression profiles
to characterize aging and interventions could be that aging
is a kind of mosaic. Different organs seem to age at different
rates, and you die from whatever the weakest link is. So you
could look wonderful based on many different markers and drop
dead the next day because of one weak link that perhaps wasn't
even measured. How do you interpret that in terms of your
biological age? Your biological health may be difficult to
quantify, let alone your biological age.
S.S.: That's right. It's not clear to me whether we'll be
able to get gene expression biomarkers from humans, for instance,
from the cerebral cortex!
L.E.: Indeed. That's a very good point. You may not have
access to the tissues you actually need to answer the question.
S.S.: But it is a promising technology.
L.E.: Yes. It's very promising technology, and as you said
earlier, you do have some alternatives to biopsying brains
and livers and so on in order to get results. So I think that
that's hopeful and promising. But have you found any contradictory
effects of calorie restriction and aging, such as maybe having
both an increase and a decrease in cell proliferation factors
or both an increase and a decrease in the tendency for apoptosis
or for inflammation? Or is everything consistent?
S.S.: We're not really looking at physiology. We're inferring
changes in physiology from changes in gene expression. There
is a lot more work to be done to see whether these changes
in physiology actually occur as a result of changes in gene
expression. Really, we're at the beginning of a very exciting
era, and I think we need to use proteomics ultimately. We're
going to need to know more about what's happening to the proteins
in the cell every time we check.
L.E.: So, as you said before, this is really a screening
process, a way of generating leads for further development.
S.S.: It's a very powerful technology. It gives us an enormous
amount of extremely useful information. It provides us with
biomarkers for drug screening. It's an unbiased assay genome-wide
of effects of caloric restriction, short and long-term. But
it is not the entire story by any means.
L.E.: Your paper was based on the liver. What is the importance
of the liver in human aging?
S.S.: The liver is an interesting organ. It is one that ages
well. There are people who, of course, die from liver disease.
That is a killer of the elderly. We were attracted to studies
on the liver because it has such a powerful influence on physiology.
One major problem that develops with aging is metabolism of
drugs. They're not metabolized as well, partially because
of loss of the enzymes that are involved in metabolism and
partially because of a decrease in blood flow to the liver
and in the volume of the liver with age. Most of the proteins
that are circulating through your body in the blood, that
aren't in blood cells, come from the liver, and the composition
of those proteins has a major impact on wound healing, on
clotting and on atherosclerosis. The composition of your blood
as far as glucose and insulin levels go is partially due to
the responsiveness of the liver to insulin, and has a major
impact on vision and on your circulatory system. So the liver
sits at a very central spot. Also, it's partially mitotic.
So far, gerontologists have been attracted very often to organs
whose cells don't divide very much. There is a good rationale
for that interest, because these organs can't create new cells
when old cells get damaged or die. But most of the tissues
in our body are able to renew themselves, and many of those
tissues that can renew themselves have major problems with
age. They have problems with being able to renew themselves
properly. So I think that they also are very important to
look at.
L.E.: Did the changes you observed actually reflect changes
in liver function that normally occur with aging?
S.S.: We found quite a number of changes that correlate well
with the known changes in liver function. We and others have
shown that there is a decline in the expression of enzymes
that are involved in drug metabolism in the liver, and that
fits very well with the loss of those differentiated (liver-specific)
functions. Other specialized (differentiated) functions of
tissues begin to decline with age, and certainly there was
a decline in genes that are important for cell division and
an increase in genes that tend to block cell division. That,
for instance, fits in very well with the loss of the capacity
of the liver with age to repair itself and to produce new
cells that replace damaged cells.
L.E.: So, some changes that seem to be related to liver aging
per se consist of the loss of liver-specific functions.
S.S.: Yes.
L.E.: Why do you think the liver does so well with aging?
You've mentioned a lot of change that takes place, but in
reality, compared to other tissues, not much aging seems to
be happening in the liver.
S.S.: It's a difficult question. There is a standard bias
that the liver ages well. This is based on physiological measurements
and endpoints. People compare how many people die of or are
afflicted with liver disease versus brain disease or muscle
disease. I think there were certainly differences between
the liver and tissues without the potential to divide. We
saw new things that we think occurred because the liver does
have the potential to divide. I don't really know the reason
for all of the tissue-specific differences. We're going to
need more data and more time to think about the data we have
before it becomes clear what's going on.
L.E.: How do the changes you do see with aging in the liver
compare to changes seen in the non-dividing tissues?
S.S.: We found, as had Weindruch and Prolla in their studies
of gene expression in brain and muscle, that the older the
animals got, the more there was expression of genes that seemed
to indicate that the animal was undergoing inflammatory stress
and other kinds of stresses. Stress gene expression and inflammatory
gene expression rose with age, and in the current study the
majority of those changes were reversed by short-term and
long-term calorie restriction, which means that even late
in life you can improve your physiology and reduce the level
of physiological stress rather dramatically by under-eating.
On the other hand, we saw a lot of action in the pro- and
anti-apoptotic genes that you do not see in the brain and
the muscle. The brain and muscle cells have to make very different
kinds of decisions about whether to commit suicide or not.
L.E.: Very interesting point.
S.S.: Yes. And we also saw genes that were either pro cell
division or anti cell division that were affected by aging
and calorie restriction. Again, these genes would not be genes
that you would expect to find changing in non-dividing tissues.
Although, interestingly, they did find a few genes that are
involved in cell division changing in the brain.
L.E.: Could that be due to the presence of glial (non-nerve)
cells or vascular cells in the brain?
S.S.: Yes, it could be, you're right. We know too that neuronal
stem cells are capable of dividing and migrating to different
regions of the brain.
L.E.: What about genes related to energy metabolism?
S.S.: We've looked in great detail at the genes that are
involved in energy metabolism in the liver and in muscle.
We find that calorie restriction has major effects on those.
And we've looked in liver and muscle on the effects of feeding
on gene expression related to energy metabolism. The results
can be summed up by saying that a calorically restricted animal,
even one that has just eaten, is rapidly turning over (renewing)
its peripheral tissues. It's an effect that you'd expect,
in fact, because insulin is a very powerful anabolic (tissue-building)
hormone. Insulin levels fall in calorie-restricted animals,
but you get a spike (rapid increase) in insulin levels after
they eat, an intense spike, and they're very insulin-sensitive
(able to respond to insulin). What I expect happens after
they eat is an intense wave of protein biosynthesis under
the influence of insulin. As soon as the insulin level falls,
they start to break down their proteins again and put the
products out into the blood for energy generation. This keeps
calorie-restricted animals constantly recycling their proteins
so they don't accumulate damaged, oxidized, old, defective
and toxic proteins.
L.E.: Has this kind of recycling been seen in liver and muscle?
S.S.: Yes. Our data in combination with the work of others
supports our general conclusion that calorie restriction causes
and maintains enhanced turnover of peripheral tissue protein
into old age, whereas turnover is lower and declines further
with age in non-restricted animals.
Continued...
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