Methylation, Epigenetics and Longevity


A new study, partly funded by the Life Extension Foundation, hopes to demonstrate how increasing the body's methylation through proper nutritional supplementation may extend life span.

The other night I was watching the movie GATTACA, a film about a future time when we are all identified by our DNA sequence. Of course, with this DNA sequence-based identity comes lots of distinctions of class, with some people given opportunities in life and others shunted to menial tasks. One of the distinctions in the movie that can be made from DNA sequence is anticipated life span, to the year.

Even today we know that DNA sequence alone only determines, at best, a broad life-span range. Most laboratory mice and rats are grown as genetically identical strains through inbreeding, yet these groups of genetically identical mice and rats die over quite a range of time. For example, most mice of the C57BL6 strain living in similar conditions will die between 18 and 33 months of age (e.g. Finch 1990, Blackwell et al. 1995). That's similar to most humans dying between 50 and 90 years of age, and humans are not even genetically identical, nor do we all eat the same food, drink the same water or live in the same environment.

The point is, if genetics called the whole tune, the C57BL6 mice should all drop dead on a Tuesday afternoon, precisely two years, three days and six minutes after their birth. So why don't they?

It has long been known that normal mammalian cells grown in laboratory culture will become senescent, stop growing and eventually die. These normal cells grown in laboratory culture dishes lose something called "DNA methylation" the more they grow and divide (Wilson and Jones 1983). In contrast, tumor cells, although low in DNA methylation to begin with, don't necessarily lose more DNA methylation as they grow and divide. The fact is, tumor cells are immortal.

Methylation is the passing of a chemical fragment called a methyl group (a carbon atom linked to three hydrogen atoms) from one molecule to another. This chemical "tag" acts as an all-important signal and structural modification throughout our bodies (Mitchell 1998). Although there are many uses of methylation, DNA methylation is one of the essential, and one of the most important, uses of methyl groups. In fact, if methylation of DNA is limited or prevented, mouse embryos won't develop and life just stops (Li, et al., 1992).

In whole animals, DNA methylation also is lost with age. In 1967, Dr. Boris Vanyushin and co-workers in Moscow described this process in salmon, and later showed that DNA methylation was lost with age in most tissues studied in cattle and rats (Vanyushin et al. 1973, Romanov and Vanyushin 1981). Likewise, scientists in the U.S. and Japan showed loss of DNA methylation with aging in many tissues of mice (Singhal et al. 1987, reviewed in Cooney 1993).

Dr. Vincent Wilson and co-workers showed that the longer an animal species' life span, or the greater the doubling potential of a cell type, the better it is able to maintain its DNA methylation. Their studies indicate that humans maintain their DNA methylation much better than do mice, and, as we know, humans live much longer than any type of mouse.

These studies raised the possibility that manipulating DNA methylation in the laboratory might alter the life span of cells or whole animals. We now know that short treatments with DNA-methylation inhibitors significantly decrease the doubling potential or "life span" of normal human fibroblast cells (Holliday 1986, reviewed in Cooney 1993).

Can methylation enhancement, in turn, increase life span?

A few years ago, the Life Extension Foundation began to recognize the broad involvement of a substance called S-adenosylmethionine (SAMe) -- an "active" source of methyl groups, a high-energy source made from ATP, our cells' "energy currency," and the amino acid methionine -- and methylation in health and longevity. Dr. Paul Frankel introduced Foundation President Saul Kent to my research on methyl supplementation and epigenetics. The Foundation agreed to support this research and together we organized a project, called Methylation, Epigenetics and Longevity.

There are several objectives of this project, including determining whether long-term dietary methyl supplementation or SAMe supplementation in rats will effect changes in longevity, in age-related pathology, and in such molecular parameters as DNA methylation, SAMe, a reaction product of SAMe called S-adenosylhomocysteine (SAH), and homocysteine, the most important biochemical risk factor for vascular disease (Frankel & Mitchell 1997). Another objective is to determine if measures of DNA methylation, SAMe, and homocysteine in the blood are indicators of longevity and can act as biomarkers of aging.

I proposed that deficiencies in methyl metabolism exist from the time animals are young. A short-lived species, such as a mouse, would have evolved a more severe inherent deficiency in methyl metabolism than a long-lived species such as a human. Importantly, these methyl deficiencies would have evolved in animals eating a balance of nutrients found in food from nature, and would therefore be at least partially dependent on the diet.

Thus, I proposed that by manipulating the diet so that the nutrients for methylation are increased, it may be possible to control these deficiencies. This is why we don't, and never did, get all the vitamins we need from our food (Cooney 1993). We need healthful longevity, yet the vitamins in our food only "warranty" most of us for youthful reproduction!

The Methylation, Epigenetics and Longevity project will test new important markers for our health, in particular blood SAMe and white blood cell DNA methylation. We know that homocysteine levels increase with age in humans (Brattstrom et al. 1994). In contrast, T-cell DNA methylation declines as we age and is likely a cause of autoimmune disease (Yung et al. 1995). How blood SAMe changes with age isn't known for sure, but when blood SAMe is low, it causes depression and is a risk factor for heart attack.

Despite all of this, tests for these conditions are not routinely done. The planned metabolic measures in our study will help relate this work to other important areas of health in addition to longevity, and something called epigenetics, which can be defined as the heritable control of the expression and use of DNA sequence.

Methylation has a huge number of effects in addition to affecting DNA methylation in normal cells, as well as cancer cells and aging cells. Low DNA methylation of blood T-cells appears to cause some lupus and rheumatoid arthritis in humans and causes autoimmune disease in mice (Yung et al. 1995). Methylation also is used to make melatonin (the "sleep" hormone), adrenaline (the fight-or-flight hormone), acetylcholine (a neuro-transmitter), creatine (for muscle energy metabolism), carnitine (involved in fat burning in mitochondria), and choline (fat mobilization and cell membrane fluidity).

Understanding methyl metabolism and the effects of methyl supplements is important for understanding the biochemical processes that affect our health. For example, methyl metabolism is essential for the metabolism and transport of fats and cholesterol, for the metabolism of several neuro-transmitters and in the action of antidepressant drugs. Blood SAMe is low in depressed humans (Alpert and Fava 1997) or after drug abuse in mice (Cooney et al. 1998). Folic-acid deficiency appears to prevent the effectiveness of certain anti-depressant drugs, including fluoxetine (the active ingredient in Prozac), and SAMe itself is an effective antidepressant in humans (Kagan et al. 1990, Fava et al. 1995, Alpert and Fava 1997).

Methylation is also important for repair of age-related protein damage in our nervous systems and throughout our bodies. In fact, this type of repair is essential for longevity in mice. If a certain SAMe-dependent protein-repair gene is disabled or "knocked out" in mice, the animals live only for about seven weeks (in a range of three to nine weeks), instead of their normal 20 to 30 months (Kim et al. 1997). In humans, this type of repair appears to be important in a number of areas, including repairs that may help prevent Alzheimer's disease and cataracts.

Maintenance of DNA methylation is not only essential to our health in the long term (in avoiding cancer and aging), but it also appears to have a daily role in maintaining our chromosomes, immune systems and in suppressing viral infections.

Like nearly all chemical reactions in our bodies, DNA methylation is accomplished using a protein catalyst, an enzyme. For DNA methylation, the enzyme is called DNA methyltransferase, which facilitates the methylation reaction by positioning SAMe and the cytosine base of DNA very close to each other. DNA methyltransferase is like a three-dimensional reusable molecular scaffold for positioning these molecules conveniently where the work needs to be done. DNA methyltransferase also uses zinc to help bind DNA, although the full role of zinc is not yet known (Bestor 1992).

Let's take a look at nutrition and methyl supplements, and how they might have an impact on the heritable control of the expression and use of DNA sequence (epigenetics).

SAMe is needed for DNA methylation and for most enzymatic methylation in cells. SAMe is found mainly inside our cells and tissues, and can readily be measured in the red cells of our blood (Wise et al. 1997). We probably get only a tiny fraction of our SAMe directly from food, but we need methyl metabolism to produce SAMe, and methyl metabolism is dependent on a dietary source of the absolutely essential nutrients vitamin B12, folates, methionine and zinc, and of conditionally essential nutrients such as choline and betaine.

These co-factors are like essential tools needed on the protein scaffold to get the job done. Methyl-transferase enzymes using SAMe are inhibited by the reaction product SAH. Consequently, the DNA methyltransferase enzyme requires SAMe, is inhibited by SAH, and also uses zinc as a co-factor. SAH is broken down to homocysteine and adenosine. Homocysteine can then be recycled by methyl metabolism to produce methionine and, subsequently, SAMe.

We know that methyl-deficient diets will cause liver cancer, vascular disease and shorter life span in animals. This was published in 1946 by Salmon and Copeland, who showed that a choline-deficient, low-methionine diet caused liver cancer in rats, and in 1954 by Wilgram et al., who showed that choline deficiency caused atherosclerosis and death in young rats. The importance of methyl groups in avoiding liver cancer was later more clearly shown and extended by Dr. Lionel Poirier and co-workers (see Poirier 1994), and the role of homocysteine in arteriosclerosis has been shown for humans as first proposed by Dr. Kilmer McCully in 1969 (see McCully 1997). (For a full explanation of McCully's contribution to the study of homocysteine, see LIFE EXTENSION magazine, November 1997.)

Scientists have caused cancer development in rats by treating them with specific cancer-causing chemicals. This causes pre-cancerous lesions in the liver that are low in DNA methylation and high in oncogene expression -- that is, use of genes that under certain conditions can cause normal cells to become cancerous. Later, these rats develop liver cancer. At least three different cancer-causing procedures have now been used to show that this progress toward cancer can be halted in most cases by extended treatment with SAMe. SAMe not only reduces oncogene expression, but also increases DNA methylation. SAMe also greatly reduces the number of animals that later develop liver cancer (Pascale et al. 1992, 1995; Simile et al. 1996).

Betaine has been shown to increase SAMe in rats (Barak et al. 1994) and mice (Wise et al. 1997), and thus both SAMe supplements and methyl supplements that include betaine make good choices in trying to prevent spontaneous cancer in rats.

Because I knew that DNA methylation was dependent on methyl metabolism, several years ago I proposed that one reason we lose methylation with age is that our metabolisms just weren't up to snuff as far as maintaining our DNA methylation, keeping our homocysteine low or handling other longevity-assuring aspects of methyl metabolism (Cooney 1993, 1994). I also proposed that most of our cells have inherent, built-in deficiencies that compromise methyl metabolism, this leads in turn to a gradual loss of DNA methylation, and that these deficiencies and their effects are a mechanism of aging. As such, they contribute to limited normal cell growth, DNA breaks, aging and cancer.

On the surface, built-in deficiencies in our DNA methylation system or in methyl metabolism seem like a cruel trick of nature. But if we look at it from an evolutionary point of view, it makes a lot of sense. Let's use mice as an example. In nature, most mice will die either of starvation or be killed by predators, disease, drought or other environmental hazards long before they are greatly affected by aging. Likewise, those mice that survive will generally reproduce long before they are greatly affected by aging.

These considerations make a long-lived mouse unlikely and unnecessary. Why? Because it takes lots resources, including energy, essential fatty acids, choline, zinc, folic acid and more to keep an animal healthy and well-maintained for a long life, and these resources would be better expended on reproduction and immediate survival early in life.

So in most animals all metabolism should be quite sufficient -- in fact, optimized -- for immediate and short-term needs, such as youthful reproduction. This is a fundamental requirement of evolution. It should come as no surprise, then, that various aspects of our metabolisms and DNA methylation machinery are not set on "healthful longevity" with an outlook of hundreds of years, but are instead set on a "just-do-it" 5-minute-to-10-year horizon.

Very recently we have shown that epigenetics during embryonic development of mice is changed by methyl-supplemented diets fed to their mothers during pregnancy. These epigenetic factors are very important for health and longevity. We showed this with yellow mice, in which an epigenetically controlled gene both affects coat color and their health and longevity. Even though these mice are genetically identical, their health -- that is, their propensity for diabetes, cancer, obesity, longevity -- varies greatly (Wolff et al. 1998).

In our new project, we will study rats not only according to their specific ages, but also by following rats periodically over their lifetimes, regardless of how long they live. One of the things We hope to learn from this is which changes actually act as biomarkers for aging and which represent survival mechanisms of special long-lived minorities.

Rats will be maintained as control, methyl-supplemented, or SAMe-supplemented groups, and several determinations will be made. Their longevity will be determined by time of natural death, and also will be monitored at a specific time-point for age-related pathology. Blood will be collected at two-month intervals from specific rats of each group and the parameters of blood plasma homocysteine, red blood cell SAMe (RBC-SAM, Wise et al. 1997) and leukocyte DNA methylation (Cooney et al. 1997) will be determined. These measures of DNA methylation, SAMe and homocysteine will be correlated with the longevity of these individuals.

These studies will use specific methyl supplements and SAMe supplements based on our prior successful studies of metabolism, DNA methylation and epigenetics in rats and mice. They should tell us how these supplements affect SAMe, SAH, DNA methylation and homocysteine, as well as a number of more common measures, such as cholesterol and glucose. They also will demonstrate how these supplements affect age-related pathology and overall longevity. And they should give us information on how the parameters might be used as biomarkers to predict length of life and, more importantly, how to increase length of life.

The recent cloning demonstrations -- such as Dolly the cloned sheep and Cumulina the cloned mouse -- show that mammals can be cloned from adult cells. This has lots of implications for those of us interested in epigenetics and longevity (Wilmut et al. 1997, Wakayama et al. 1998).

The ability to clone almost certainly means that DNA sequence is unchanged from embryonic cells to those adult cells. This underscores that it is largely epigenetics that makes up the "thin blue line" between normal cells and cancer and aged cells. Even as cloning and related techniques make it easier to produce organs, bodies or stem cells, you still need to maintain the epigenetics of tissues and cells; otherwise these will suffer the same fate as the originals. These new discoveries in cloning emphasize that epigenetics is our next big frontier.

The cloning of animals is not just important to our basic understanding of biology and to practical advances in longevity research. It also is an inspiration to those who know that a vastly longer, healthy life is attainable. Ian Wilmut and his team, the cloners of Dolly the sheep, simply didn't believe the many scientists who said that "you can't clone mammals from adult cells." Likewise, those who say we can't live past a certain life span, or that supplements don't improve health, will need to reevaluate their suppositions or risk being left far behind.

We have in front of us a great frontier for supplement research and great opportunities to improve our health and lengthen our lives. Because many of us have been taking supplements for years, epidemiologists now have been able to prove that supplements have health benefits. But this is only the beginning. Optimal levels and optimal combinations of supplements are not known because supplement research has been neglected in the past. Supplement research will now expand greatly, and holds enormous promise to vastly improve our health, well being and longevity.

Further Reading

Rev 55, p145-149. Barak AJ, Beckenhauer HC, Tuma DJ (1994) S-adenosylmethionine generation and prevention of alcoholic fatty liver by betaine. Alcohol 11, p501-503.

Bestor TH (1998) The host defence function of genomic methylation patterns. Novartis Found Symp 214, p187-195.

Bestor TH (1992) Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. EMBO J 11,2611-2617.

Blackwell BN, Bucci TJ, Hart RW, Turturro A (1995) Longevity, body weight, and neoplasia in ad libitum-fed and diet-restricted C57BL6 mice fed NIH-31 open formula diet. Toxicol Pathol 123, 570-582.

Brattstrom L, Lindgren A, Israelsson B, Andersson A, Hultberg B (1994) Homocysteine and cysteine: determinants of plasma levels in middle-aged and elderly subjects. J Intern Med 236 p633-641.

Cooney CA (1993) Are somatic cells inherently deficient in methylation metabolism? A proposed mechanism for DNA methylation loss, senescence and aging. Growth Der Aging 57, p261-273.

Cooney CA (1994) Methylation metabolism has a central role in mammalian longevity. AGE 17, p166-167.

Cooney, C. A., Wise, C.W., Poirier, L.A. (1997) An Improved Sample Preparation Method for the Quantitative HPLC Determination of 5-Methyldeoxycytidine in Animal Tissue DNA. J Liq Chrom 20, p1279-1293.

Cooney CA, Wise CK, Poirier LA, Ali SF (1998) Methamphetamine treatment affects blood and liver S-adenosylmethionine (SAM) in mice. Correlation with dopamine depletion in the striatum. Ann N Y Acad Sci 30, p191-200.

Counts JL, Sarmiento JI, Harbison ML, Downing JC, McClain RM, Goodman JL (1996) Cell proliferation and global methylation status changes in mouse liver after phenobarbital and/or choline-devoid, methionine-deficient diet administration. Carcinogenesis 17, p1251-1257.

Counts JL, Goodman JI (1995) Hypomethylation of DNA: a possible epigenetic mechanism involved in tumor promotion. Prog Clin Biol Res 391, p81-101.

Fava M, Giannelli A, Rapisarda V, Patralia A, Guaraldi GP (1995) Rapidity of onset of the antidepressant effect of parenteral S-adenosyl-L-methionine. Psychiatry Res 56, p295-297.

Finch CE (1990) Longevity, Senescence and the Genome. Chicago: Univ. Chicago Press.

Frankel P and Mitchell T (1997) Homocysteine. Homocysteine and heart attacks. Life Extension July 1997 p8-17.

Holliday R (1986) Strong effects of 5-azacytidine on the in vitro lifespan of human diploid fibroblasts. Exp Cell Res 166, p543-552.

Kagan BL, Sultzer DL, Rosenlicht N, Gerner RH (1990) Oral S-adenosylmethionine in depression: a randomized, double-blind, placebo-controlled trial. Am J Psychiatry 147, p591-595.

Kim E, Lowenson JD, MacLaren DC, Clarke S, Young SG (1997) Deficiency of a protein-repair enzyme results in the accumulation of altered proteins, retardation of growth, and fatal seizures in mice. Proc Natl Acad Sci U S A 94, p6132-6137.

Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915-926.

McCully K (1997) The Homocysteine Revolution. Keats Publishing, New Canaan CT.

Millian NS, Gatrow TA (1998) Human betaine-homocysteine methyltransferase is a zinc metalloenzyme. Arch Biochem Biophys 356, p93-98.

Mitchell T (1998) Methylation. A little known but essential process. Life Extension. August 1998 p23-26.

Orentreich N, Matias JR, DeFelice A, Zimmerman JA (1993) Low methionine ingestion by rats extends life span. J Nutr 123, 269-274.

Pascale RM, Simile MM, De Miglio MR, Nufris A, Daino L, Seddaiu MA, Rao PM, Rajalakshmi S, Sarma DS, Feo F (1995) Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid. Carcinogenesis 16, p427-430.

Pascale RM, Marras V, Simile MM, Daino L, Pinna G, Bennati S, Carta M, Seddaiu MA, Massarelli G, Feo F (1992) Chemoprevention of rat liver carcinogenesis by S-adenosyl-L-methionine: a long-term study. Cancer Res 52, p4979-4986.

Poirier LA (1994) Methyl group deficiency in hepatocarcinogenesis. Drug Metab. Rev. 26, 185-199.

Romanov GA, Vanyushin BF (1981) Methylation of reiterated sequences in mammalian DNAs. Effects of the tissue type, age, malignancy and hormonal induction. Biochim Biophys Acta 653, p204-218.

Simile MM, Saviozzi M, De Miglio MR, Muroni MR, Nufris A, Pascale RM, Malvaldi G, Feo F (1996) Persistent chemopreventive effect of S-adenosyl-L-methionine on the development of liver putative preneoplastic lesions induced by thiobenzamide in diethylnitrosamine-initiated rats. Carcinogenesis 17, 1533-1537.

Singhal RP, Mays-Hoopes LL, Eichhorn GL (1987) DNA methylation in aging of mice. Mech Ageing Dev 41, p199-210.

Vanyushin BF, Nemirovsky LE, Klimenko VV, Vasiliev VK, Belozersky AN (1973) The 5-methylcytosine in DNA of rats. Tissue and age specificity and the changes induced by hydrocortisone and other agents. Gerontologia 19, p138-152.


The Life Extension Foundation already has contributed $18,000 to Dr. Craig Cooney's research. However, contributing the balance of what is needed has been difficult. Once again, much of our resources have been diverted to fighting legal battles to protect individual health rights. Since 1987, the harassments of government bureaucracies have repeatedly diverted funding intended for critical research that could radically extend life span. We thank members for participating in protests against the FDA and other government agencies whose illegal actions pose a serious threat to our right to exist.

The Life Extension Foundation supports scientific research primarily through membership dues and product purchases. While we continue to fund ongoing research projects, we cannot all provide the funding Dr. Cooney needs now to start his project. As a result, we are asking for donations from members.

We have set up the Cooney Research Project, and ask that members who would like to see this important research commence send donations payable to the Life Extension Foundation, P.O. Box 229120, Hollywood, Fla., 33022. Or, phone in your donation by calling 1-800-544-4440.

Please indicate that your donation is for the Cooney Research Project. All donations we receive for the next several months will support this research that could lead to a major breakthrough in the fight against aging.

--The Foundation

Life Extension Foundation.


By Craig Cooney

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