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LE Magazine December 2001
Page 4 of 4
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, on atherosclerosis, etc. 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 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 do 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?
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| Dr. Spindler identifies where regulatory proteins bind to a specific gene. | |
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.
L.E.: Might this explain why there is an increased inflammatory response with aging and why calorie restriction tends to reverse that?
S.S.: I don't know what that inflammatory response is due to, it's very intriguing. Is it a physiological inflammation, or is it inappropriate changes in the regulators of those inflammatory genes that are not really related to true inflammation?
L.E.: In other words, perhaps the body thinks that there is an enemy that doesn't really exist.
S.S.: Or is there real inflammation that they're responding to, and if so, what's the inducer? It's a very interesting and complex question, and something that's going to be exciting as more people begin to work on it and try to deduce really what's going on.
L.E.: Well, we certainly know from the proponents of the glycation hypothesis that macrophages (inflammatory cells) will attack glycated myelin and so forth because of the molecular change involved in glycation. There could also be other, analogous changes that would accumulate simply by failure of turnover of the proteins involved.
S.S.: We actually made sections and had them examined by the Pathology Laboratory at U.C.L.A. We didn't see any signs of increased macrophage activity in the calorie-restricted livers, or in the old livers versus the younger livers. But those studies were just preliminary and were just quick looks. I think there's a lot more work to be done to investigate that phenomenon.
L.E.: In comparing changes that you have seen and other people have seen in gene expression from tissue to tissue, can you draw any conclusions about the nature of aging in general?
S.S.: Well, so far, based on aging in brain and muscle and liver, a generalization is that aging seems to involve an increase in inflammatory gene expression and an increase in what's broadly categorized as stress gene expression. Caloric restriction seems to reverse most of that. I think that's our major consistency right now.
L.E.: The stress proteins, meaning certain proteins that stabilize other proteins against environmental stresses, were one of the major protein classes that were suggested to rise with aging in the first Weindruch and Prolla gene profiling experiment. Much was made of that observation.
S.S.: Well before the Weindruch and Prolla papers came out, we were publishing papers that showed that these stress proteins, or "chaperones," go down with caloric restriction.
L.E.: Quite right, and that they go up with aging.
S.S.: Yes. Heat shock proteins are chaperones. Chaperones respond to stress. You have to understand the purpose of chaperones to understand why a calorie restriction mediated decrease in chaperone levels may be extremely important.
Normal proteins have to have a certain shape in order to work, and stress may cause "unfolding" of proteins so that they lose their proper shape. Chaperones are required at a certain level to assist with or correct folding of proteins. They also go around and rescue proteins that have become improperly folded and refold them properly. If they can't be refolded, the chaperones tag them with ubiquitin so they can be degraded and eliminated from the cell.
When you have a stress, like, for instance, exposure to radiation, the cells that are damaged have to make a decision. Are they going to repair themselves and, if they do, has the repair been successful? Or has the damage been too severe, such that the cell, when it repairs itself, is still damaged and may become a cancer cell? Or might the cell secrete destructive local hormones to the cells around it? In such cases, the cell needs to make the appropriate decision to commit suicide and be eliminated from the body and replaced by a healthy cell.
These kinds of molecular decisions are being made all the time, not just after a dose of radiation. As part of the normal process of living, for example, there is damage to our DNA that has to be repaired, and molecular decisions have to be made about how to institute and evaluate the repair process.
Chaperones are also involved in the decision about whether a cell will commit suicide. Chaperones are a part of the machinery for regulating gene expression in some key instances. For instance, high chaperone levels inhibit apoptosis.
L.E.: Apoptosis being cell suicide or the elimination of defective cells such as pre-cancerous cells.
S.S.: That's right. Some chaperones prevent apoptosis, and if you lower the levels of those chaperones, then you encourage more apoptosis.
So, if you have high chaperone levels with age, that tends to make cells less apt to commit suicide and more apt to continue to exist, even when they're severely damaged and may be secreting harmful cytokines to the tissues around them, or possibly converting to a cancer cell. So, when calorie restriction lowers those chaperone levels, it releases some of those factors slightly more, so the decision is slightly more likely to be made to commit suicide and kill those damaged cells that might otherwise survive.
Therefore, calorie restriction is anti-cancer. That's well established. Calorie restriction is also pro-apoptotic. It promotes cell suicide of damaged cells and we think the reason it's pro-apoptotic is because it lowers the chaperone levels.
L.E.: I suppose one might imagine that over-expressing chaperones could be positive, say in a post-mitotic tissue, in which dead cells can't be replaced. But then again, you have to balance cell loss against the probability of dying very rapidly from cancer.
S.S.: That's right, and those are the kinds of complex and critical decisions that cells are making in our body all the time.
L.E.: The inflammatory proteins that you saw going up are kind of a motley crew, are they not? And a lot of inflammatory proteins that might be most informative about what's going on don't seem to be there.
S.S.: That may be partly a function of which genes were on the chip and partly a function of the subtleties of how proteins work together. If the level of one of the components of a group of proteins changes, whether that has an effect on overall activity and whether that will influence other specific proteins may not be obvious. I'll just give you an example. One of the chaperones, HSP-90, does a lot of things. It binds to some steroid hormone receptors and keeps them out of the nucleus and inactive. When the hormone that interacts with the receptor shows up, it disassociates the receptor from the chaperone and the receptor now becomes active. That same HSP-90 also binds to two pro-apoptosis factors.
L.E.: So there can be multiple effects of a given interaction.
S.S.: Yes. HSP-90 also is only one of a group of proteins with a relationship to steroid hormone receptors or with the pro-apoptotic factors. So, there's a great deal left to be found out about how all of these specific gene changes affect physiology.
L.E.: Yes, it's very complicated.
S.S.: I hope the leads that we're getting will attract people to investigate these kinds of questions.
L.E.: Yes. Basically, you're trying to understand the changes that take place over time in a system that nobody even understands to start with.
S.S.: It's an immensely complicated system, but not infinitely complicated. I'm confident that we'll be able to understand it. It's just going to take time and work.
L.E.: And in fact you've already identified a lot of pathways that have implications for the control of disease, so you're certainly making rapid progress. But progress can sometimes bring confusion, too. For example, your data and a lot of other observations indicate that calorie restriction reduces growth hormone function, thyroid hormone function and cell proliferation, and yet we need these functions in order to live. Can you make anything out of that, yet, at this point in the game?
S.S.: There's more complexity, but the functions that we need to procreate our species are not always the same as the functions we need to survive a long time. There are hormonal systems at work that want to speed us to fertility and fecundity and ensure that we'll reproduce. There are also imperatives to ensure that we'll produce offspring that will be aggressive for men, or that will be nurturing for women. Those are a different set of priorities than living a long time. In fact, calorie restricted animals are not very good at procreating, so it may be that some of these effects that we're seeing seem to be counter-intuitive because we're not keeping in mind what it is they're trying to accomplish.
L.E.: It seems that several genes whose expression changed the most with aging were not affected by either short-term or long-term calorie restriction.
S.S.: Right, and that's not to say that those changes with aging are not important, because of course calorie restriction does not stop aging. It just slows it or reverses it to an extent.
L.E.: Indeed. Do you have any plans to go beyond calorie restriction and CR mimetics and to attack those aspects of aging that persist in the face of calorie restriction?
S.S.: Not anytime soon. The challenge of finding out what causes calorie restriction to extend life span is challenging enough for now.
L.E.: Dr. Spindler, thank you very much for your interview.
S.S.: Thank you.
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