Aging with Dr. Tory Hagen

Tory Hagen, Ph.D.
Assistant Professor of Biochemistry & Biophysics
Linus Pauling Institute Principal Investigator

The following is a question and answer session between the editor and Dr. Tory Hagen.

Q: Your research has focused on oxidative stress and aging. What is oxidative stress and what role does it play in the aging process?

A: We are very interested in experimentally testing the so-called "free radical theory of aging". This theory, one of over 300 theories advanced to explain aging, states that our cells are constantly bombarded by free radicals, and that some of these free radicals, (or oxidants) can evade the vast array of antioxidant defenses and damage important biological molecules, such as DNA. Some of this damage would be expected to accumulate over our life-span and eventually cause cell and organ dysfunction, a loss in the ability to respond to environmental stress, age-related diseases (cancer, Alzheimer's disease, or cardiovascular problems) and ultimately lead to death of the individual. This theory is compelling because it encompasses so many of the things that we know to occur, both in our cells and during aging. For instance, we know that we must constantly consume oxygen in order to carry out normal cellular metabolism. On a cellular basis, oxygen is mostly used in organelles called mitochondria. These organelles have been called our cells' "power-plants" because this is where raw fuels that we eat (proteins, carbohydrates, and fats) are converted into a useful form of energy (adenosine tri- phosphate, or ATP) that the cell uses to carry out necessary chemical processes. This conversion process requires oxygen, which is used by the mitochondrion as an "electron acceptor". Consequently, this vital energy production would grind to a halt if we did not constantly breathe oxygen. Even though oxygen consumption is vital for our very survival there are unfortunate consequences for using oxygen. Oxygen can be converted into very deleterious forms called reactive oxygen species (ROS), which include free radicals, the same compounds that can be formed during a nuclear explosion. It now appears that ROS constantly arise as by-products of normal metabolism. Thus, using oxygen as a central part of our metabolism is really a double-edged sword-we need it to provide energy to carry out normal metabolic processes, but we are also constantly converting some of the oxygen that we breathe into ROS that damage important biomolecules and eventually lead to cell and organ dysfunction.

Q: Do you think aging then might be due partly to improper or incomplete repair of some of this damage caused by free radicals?

A: Researchers have observed that certain components of the cellular "house-keeping machinery", including repair systems, tend to decline with age. Therefore, it appears to me that aging affects key components that significantly increase oxidative stress. First, cells don't seem to handle oxygen as efficiently, which increases free radical production. Second, our antioxidant defenses decline, and, finally, the repair systems tend to decline. All this results in greater oxidative stress to our bodies, leading to a vicious cycle of more oxidative damage that, in turn, results in loss of cell functions.

Q: There has been a lot of media attention recently on telomeres and the role they may play in aging, cancer, and other pathologies. What is your opinion of the role of the telomere in the aging process?

A: As a scientist, I was initially intrigued with these findings. Telomeres are the ends of the chromosomes that shorten when the cell divides. There has been speculation that after a certain number of cell divisions telomere loss is so acute that the cell can no longer divide and then it dies. This suggests that we have a finite number of cell divisions before the organ is no longer able to renew itself. However, if you look at this phenomenon a little more closely, some problems with this theory become apparent. For instance, the cells in many of our organs simply do not divide. There are tissues in our bodies called "post-mitotic", such as the heart and brain, in which cells quit dividing after development. Does that mean that our brains and hearts are senescent before we reach puberty? I don't think so!

Q: Could one examine this issue experimentally by comparing rates of aging in different organs or tissues within an organism?

A: Well, that has been tried, but it is a very difficult experiment to do. Not all cells of a given organ are dividing at any one time, so you have a very small number of cells that are dividing. How do you follow that one cell amidst the millions of other cells in order to observe how many times it divides? While the telomere theory of aging has some adherents, there are more scientists who think that the telomere may be a very important phenomenon in cancer, which is a subset of aging, but not a fundamental mechanistic explanation for aging. It certainly could not explain aging for all tissues.

Q: How does the concept of telomere truncation fit in with the so called Hayflick number, which was described by Leonard Hayflick as the finite number of cell doublings that will occur in cell culture before cells senesce and die?

A: To some extent, the telomere theory has resurrected the Hayflick model. Hayflick observed that normal cells in culture will divide for a certain number of times and then stop. Although this is very reminiscent of telomere loss, it is not exactly the same phenomenon. First of all, tissue culture conditions do not mimic what happens in a living animal. Secondly, a number of changes occur in cultured cells that are independent of telomere truncation that could result in the same phenotypic phenomenon of loss of cell division. So this phenomenon is called "replicative senescence", which does not adequately mimic all the varied changes that occur in our body during aging.

Q: Is this why you have chosen to study whole organisms, such as rats, and intact cells from their organs to understand the cellular and molecular changes that occur in aging?

A: Yes. This is why we rely on animal models to study aging. Compared to cell cultures, it's a much more difficult experimental system to use, but we get a better glimpse as to what is actually occurring in our cells as we get older. Presently, we are using rodent models to carry out our work because a lot is known physiologically about these animals, and they mimic what happens to humans as we age. An old rat is about 21/2 to 3 years old, so we don't have to wait too long to get results. We can test rather easily how micronutrients affect the "health span", as well as the life span, of an animal. By doing so we hope to gain insights into what's happening in humans.

Q: What do you mean by health span?

A: Health span is a term that describes how long a person is healthy, enjoying life, doing what one wants to be doing thanks to good health. This is in contrast to "life span", which refers to how long a person lives.

Q: Health span sounds very similar to a concept Linus Pauling promulgated many years ago. He was interested in extending the period of healthfulness in an individual. Once a population reaches the end of its life span, the death rate would be fairly rapid- not a gradual deterioration over a long period of time.

A: Yes, indeed. Sociological studies have found that, given a choice to live longer with serious illnesses or live a shorter life and maintain health and vitality as a trade-off, people tend to opt for the latter. If there was a third option of living longer and healthier lives, I think that we all would choose that!

Q: In your experimental work on rats, you've done a lot of work with liver cells. What made you select the liver as an organ of special interest?

A: The liver is a good model in some important respects and in others it is not so good. We use the liver as an "anchor organ" so that we can compare and contrast its age-related changes to those in other organs, such as the heart and the brain. The liver is a very large organ, so there is a lot of material to use in order to do many concurrent experiments. This allows us to build up a good profile of the age-related changes that occur in this vital organ. We can test the free radical theory of aging more easily than in other, smaller organs of the body. The liver is the major detoxification organ, and liver cells can still divide. Therefore, we can compare them with "post-mitotic" tissue that no longer divides, such as the heart and the brain.

Q: What differences in liver cell function do you see during the aging process?

A: In general terms, we and other investigators have found that the aging process causes us to lose the ability to respond to environmental stresses. This includes oxidative stress (free radicals) or other types of stresses like toxin exposure. We can place different types of stresses on the liver to see how these cells respond. We are also interested in doing this in other organs. For instance, the heart is exquisitely sensitive to energy status, therefore we can place a burden on the heart by simply making it beat faster.

Q: What kinds of experiments have you carried out with rat liver cells to assess differences between young and old rats and how they respond to environmental stresses?

A: One of our premises is that the mitochondria become more dysfunctional as we age. As I mentioned before, the mitochondria are specialized organelles in the cell that convert raw fuel from our diet into a usable form of energy currency, ATP. We have observed that this conversion process in old animals is no longer very efficient. The mitochondria are not able to utilize raw fuels, especially fatty acids, very well. This causes the cell to try to compensate for mitochondrial decay. This compensation may take the form of neglecting some cell functions in order to maintain more vital systems that the cell must maintain to survive. This is what we've been looking at very closely in the liver cell and in the liver as a whole. We have found that, due to mitochondrial decay, overall cellular metabolism slows. This may be the cell's attempt to compensate for mitochondrial decline. We have examined how this metabolic decline affects the cell's ability to cope with everyday stresses by using a toxin called tertiary butylhydroperoxide to determine whether cells from old animals are more susceptible to toxic insult. We recently showed that cells from old animals are twice as susceptible to this toxin as cells from young animals. Since this toxin specifically targets the mitochondria, we can now see that age-related mitochondrial decay may make us much more prone to a variety of toxicological and environmental insults. Of course, damage to the mitochondria by toxins also affects their ability to produce energy.

Q: You've described a number of problems associated with mitochondrial dysfunction or increased mitochondrial damage due to exposure to toxins. What kinds of strategies have you found that can either prevent or reverse some of these types of insults to the mitochondria?

A: Our goals are not only to understand the extent and precise nature of mitochondrial dysfunction with age and how that relates to the free radical theory of aging, but also to understand how dietary micronutrients may either mask, maintain, or improve mitochondrial function in the aging animal. We want to relate these dietary effects to the health span of animals and, hopefully, to humans. We are trying to identify the micronutrients that directly affect mitochondria. In order to do this we first have to understand what micronutrients decline with age and how this decline affects the mitochondrial function. We have found several that decline, including carnitine, which is an amino acid that transports fatty acids from the diet into the mitochondria to be converted into ATP. Carnitine levels decline by more than 50% in old animals compared to young animals. Therefore, the mitochondria are starved of dietary fuel because it simply can't be brought in to be burned. We have also found that a phospholipid-cardiolipin -declines dramatically by about 50% in old animals. Cardiolipin is very important for overall maintenance of mitochondrial function. We've also observed that mitochondria have lost certain antioxidants to a significant degree, including glutathione and vitamin C. The decline in both of these antioxidants is more pronounced in the mitochondria than in the rest of the cell. This tells us two things: we a have loss of micronutrients like carnitine and cardiolipin that can directly affect mitochondrial metabolism and vital antioxidants are lost. This means that mitochondria may be even more vulnerable to free radical insult. What can we do about this? Well, when we feed animals a compound called acetyl-L-carnitine (a more bioavailable derivative of carnitine also called ALCAR), their cells are repleted with this important substance. This results in dramatic metabolic effects. For example, many of the indicators of mitochondrial dysfunction disappear after only one month of dietary ALCAR supplementation. Metabolically, the animals are much more active, they have improved short-term memory as assessed by special tests, and we see an improvement in their overall physical activity. We see a very significant improvement in a number of metabolic parameters that would lead us to believe that these animals are simply doing much better on the supplemented diet. We have also attacked the problem associated with increased mitochondrial performance in the old animals-an increased oxidative insult to the mitochondria from oxygen free radicals-by providing a relatively obscure antioxidant called lipoic acid to the animals in their diet. Lipoic acid is a naturally occurring antioxidant found in green leafy vegetables, as well as in meat, that is required for the production of energy from glucose. Hence, lipoic acid can directly affect metabolism and in its free form is a very potent antioxidant, even more powerful than vitamin C. Lipoic acid also "spares" vitamin C and other antioxidants. We fed lipoic acid to rats and found that it can completely reverse the age-related loss of vitamin C and glutathione, an important endogenous cellular antioxidant. So lipoic acid not only acts as an antioxidant in its own right, it also repletes very important antioxidants like vitamin C. We are now exploring these functional mechanisms, which are quite complex. We think that lipoic acid may improve the uptake of vitamin C into tissues as well as increase glutathione synthesis in cells.

Q: Dr. Ewan Cameron, the Scottish surgeon who collaborated closely with Linus Pauling for many years on the clinical use of vitamin C in cancer patients, found that many of his terminal patients suffering from severe lassitude who were given doses of vitamin C reported an increased sense of well-being and had more energy. When Cameron looked into this more closely he realized that vitamin C is required for the hydroxylation reaction that converts the dietary amino acid lysine to carnitine. 

A: The loss in vitamin C may actually be causing the loss of carnitine. And we'd like to explore that in the future.

Q: How applicable are your results with liver cells to other tissues or organs in the aging rat and to people?

A: That's a good question. We've just been awarded a large NIH grant to explore these phenomena in heart function as well. We are rapidly setting up and doing these experiments. Again, in the heart and in the brain, we see a very marked age-related decline in vitamin C status. We are also seeing changes in mitochondrial function to a significant degree. So it looks like the phenomena we saw in the liver is almost a universal effect of aging. There are some differences in the rate of these changes in different organs, but it is clear that this is a general phenomenon of the aging process. Most importantly, whenever we do our dietary intervention studies with these micronutrients, we not only see effects in the liver but also systemic effects. When we put both ALCAR and lipoic acid into the diet, we also see an enhancement of short-term memory and cognitive function. That leads into the last part of your question. I think that these supplements may have an important health potential for humans not only in terms of energetics and vitality, but also as a way to slow short-term memory loss, frequently called mild cognitive impairment. Since we don't have any human clinical data yet, we can't make any claims. 

Q: Is your strategy to provide sufficient information on the molecular and biochemical functions of some of these molecules in animals that would provide a platform for human studies? 

A: Absolutely. What we are trying to do right now is understand exactly how these micronutrients work in the body. We need to understand both the proper effective dosage and whether they are completely safe. I should add that we have only done short-term feeding studies, and the benefits may or may not be preserved in long-term studies. Micronutrients can powerfully affect overall metabolism. Before going on to human clinical trials, we feel compelled to understand the ramifications of supplementation, including any potential safety problems with the use of these supplements. Therefore, I cannot recommend that people use them until this information is available.

Q: Many safety reviews on vitamin C have been published over the years, and to date there doesn't seem to be any significant problem with large doses of vitamin C, except for perhaps in certain populations, such as those with hemochromatosis. Hasn't lipoic acid been used to treat diabetic neuropathy in Europe for some time? 

A: It's been used for 40 years as a treatment for type II diabetes in Germany in relatively high doses, but the pharmacokinetics and the safety data are not widely distributed-they are primarily held by pharmaceutical companies. I have not heard of any untoward effects of lipoic acid or ALCAR or carnitine. Carnitine is naturally made in our bodies and is also a component of red meat, and lipoic acid is found in green leafy vegetables, so we routinely ingest these substances in small amounts.

Q: Are there any human diseases that might be exacerbated by these substances?

A: Lipoic acid has been reported to cause hypoglycemia if not taken with food. 

Q: Dr. Roy Walford and others have popularized the idea of extending life span by caloric restriction. How does the concept of caloric restriction fit in with some of these other ideas that we've been talking about? 

A: Actually it fits in very well. Caloric restriction is a very harsh form of food restriction in which approximately 60% of the calories is restricted. Even though this diet is severe, the life span of an animal can increase by 30 to 40%. That means that humans would live 140 to 150 years! These animals are not nutritionally restricted or malnourished -they have everything they need for proper cell function. However, it seems that food restriction orients the body to be extremely efficient. Caloric restriction also seems to maintain mitochondrial function much longer. The mitochondria become very efficient in utilizing oxygen, so there are not so many free radicals being generated. The general housekeeping machinery of the cell, the ability to respond to stresses of all types, and hormone levels are also maintained. Frankly, the major problem with caloric restriction is that it's impractical and unappealing. To achieve effects on life span similar to those seen in animals would require a very severely restricted diet-something that most people would not tolerate. Our research is designed to see if we can bring about much of the same phenomena of increased life span and health span that caloric restriction produces, but doing it instead by maintaining overall micronutrient levels. This is an example of orthomolecular medicine as defined by Linus Pauling. We want to give the right substances in the right amounts. Nutritional needs change throughout life. Some nutrients that young people normally get very well in the diet or make endogenously are simply not present to the extent needed in older people. 

Q: Can you provide us with other tantalizing glimpses about recent results and the work you plan to do in the near future? 

A: Well, as I said, we've been very pleased to have been awarded a large NIH grant to study the metabolic effects of lipoic acid and carnitine on heart function. I'm also quite gratified that we have attracted a large number of people into the lab in order to pursue our goals. So our lab and procedures are really set up and working well. Currently, we are looking at the metabolic effects of carnitine and lipoic acid in hearts and heart tissue from animals. So far, our results are similar to or better than what we observed in our liver studies.

We have another completely serendipitous finding. It's been known that there is a large age-related increase in metal accumulation, especially iron, in our tissues. In some cases, this may be harmful because free iron isn't used by the body very well and may help produce free radicals. In particular, Alzheimer's and Parkinson's patients have higher levels of accumulated iron than people who are undergoing normal aging. We have found that lipoic aid supplementation reverses this increase in unbound iron. This opens the possibility that lipoic acid may be able to lower metal-related oxidant stress and slow the progression of Alzheimer's disease. 

Q: One last question concerns what many people have called the "antioxidant network". Some of the antioxidants that you've discussed we can obtain dietarily. Other antioxidants are enzymes that are made within the body, for example, superoxide dismutase and catalase. Jim Fleming at the Linus Pauling Institute of Science and Medicine in California worked with fruit flies, which are composed of post-mitotic cells. He and his colleagues were able to increase the amount of some of these endogenous antioxidant enzymes in flies, which resulted in increased resistance to stress as well as increased life span. Do you have any concluding thoughts about how these dietary or endogenous antioxidants collaborate? 

A: You are absolutely right. You can never talk about one compound in isolation when you are dealing with something as complex as a living cell or organism. Every one of these antioxidants—glutathione, vitamin C, vitamin E, lipoic acid, uric acid, antioxidant enzymes—works in conjunction. A deficiency in one may be compensated for by another. Our work suggests that with aging, there is a true loss of this antioxidant networking. Aging causes a breakdown of this whole important web with serious consequences to biomolecules, cells, and whole organisms, which then become more vulnerable to free radicals and other environmental stresses.

Last updated November, 2000

Honoring a Scientific Giant with Nutritional Research Toward Longer, Better Lives

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