Nutritional Supplements May Help Produce a . . .
Life Extension Ring

One Ring to rule them all, One Ring to find them,
One Ring to bring them all and in the darkness bind them . . .
- J. R. R. Tolkien

The ring . . . ever-powerful, ever-present . . . has been an enduring symbol in world mythologies. Long before the pyramids of Egypt, or the gardens of Babylon, there was a tradition of ring-quest tales. Even after the rise and fall of the golden ages of Greece and Rome, the idea of the ring as a powerful metaphor lived on. Beyond the demise of the pagan gods and the ascendency of Buddha, Mohammed, and Christ, there was the ring.

Today we know the lore of the ring from Wagner's opera cycle, The Ring of the Niebelung, and the trilogy by Tolkien, The Lord of the Rings (soon to be a major motion picture). But we also know, and are learning more each day, about another ring, more powerful than those of antiquity or mythology, because it dwells in every cell of our body and, like the Holy Grail, may hold the secrets of life, to wit:

We dance round in a ring and suppose,
But the Secret sits in the middle and knows. 
- Robert Frost

Frost's poem is suggestive. Despite the passing of 64 years since Hans Krebs's discovery of the citric acid cycle - a "ring" of metabolic processes also known as the Krebs cycle (see Figure 1) - our knowledge is incomplete.¹ In the mitochondria, those "energy factories" embedded in every cell in your body, the Krebs cycle is busy at work producing ATP, the universal energy-transfer molecule, for your cellular metabolism. Through its unceasing activity, the Krebs cycle is the principal means by which our cells produce energy.

Figure 1.
The Krebs cycle is a series of enzyme-catalyzed reactions,
three of them involving NAD, that transform chemical energy stored
in the foods we eat into energy in a form that cells can use.
The ultimate product, although it does not appear in the Krebs cycle itself,
is the energy-rich molecule ATP.

RINGS OF ROMANCE
So the ring of which we speak, the "Krebs ring," is connected to an externality - it thus spins off an unexpected benevolent power - in much the way as the rings portrayed by Richard Wagner or J. R. R. Tolkien work. Their rings were sought so that the bearer could exercise power over others, while the golden rings of Grail romance - also rendered into opera by Wagner (in Parsifal), but without the spirit of the tradition from which they arose - represent love and enlightenment. They are rings of self-empowerment that liberate rather than inhibit the power of others.²

RINGS OF SHIVA
Within the Krebs cycle are certain molecules containing ring structures (very common in organic chemistry); these molecules participate in the continuous process of energy manufacture and utilization, much the way the Indian god Shiva is represented: as both creator and destroyer.

One such Shiva-like molecule is NAD (nicotinamide adenine dinucleotide - see Figure 2), which is among the most important coenzymes in the cell (coenzymes are molecules that collaborate with certain enzymes to allow them to perform their catalytic function). The nicotinamide coenzymes (there are several different forms) are electron carriers. They play an essential role in a variety of enzyme-catalyzed oxidation-reduction reactions, which are the basis for most of the body's energy-generating processes.

Figure 2.
Nicotinamide adenine dinucleotide (NAD), the molecule that facilitates
gene silencing by the Sir2 protein. The portion shown in purple is nicotinamide.

For example, NAD figures prominently not just in the Krebs cycle, but also in glycolysis, an 11-step process by which glucose is converted to lactic acid and ATP.

NAD AT LARGE
Beyond these vital functions of NAD, there is more to its utility, such as the recent, monumental discovery of its ability to break the link between the cellular metabolic rate and the pace of aging in the organism (see The Supercoiled Theory of Aging). In collaboration with a protein called silent information regulator no. 2 (Sir2p), NAD has been found to be absolutely necessary to prevent the release of ring-shaped, "destroyer" DNA fragments that, in the aging process, proliferate inside cells, to the point of cell strangulation and death. These fragments are called extrachromosomal rDNA circles, or ERCs (see Figure 3). Although NAD is a frequent enzyme cofactor in reactions such as those of the Krebs cycle, this is the first example in eukaryotes (organisms whose cells contain a distinct, membrane-bound nucleus, with its DNA arranged in chromosomes) in which NAD acts as a catalyst.

Figure 3.
The process by which extrachromosonal rDNA circles (ERCs)
form and multiply. (a) The replication of a DNA molecule
 is blocked at the fork where replication occurs. (b) One of the new
DNA strands breaks off. (c) The broken strand loops onto itself,
 forming (d) an ERC, which starts the "aging clock." As successive cell
divisions occur, the ERCs multiply but tend to remain concentrated in the
"mother cell," which eventually dies.

MAXIMUM LIFESPAN EXTENSION
The reward for understanding the mechanisms of the NAD/Sir2p collaboration well enough to take practical action is the ultimate goal of serious health seekers: maximum lifespan extension. When NAD and Sir2p join forces, the potential (or actuality) for the runaway growth of ERCs is inhibited. This mechanism is thought to be similar to that of caloric restriction, which to date has been the only known means of extending maximum lifespan in rodents, primates, and possibly humans.

While there are several candidate explanations for the mechanism of caloric restriction, including the excess oxidation-metabolism hypothesis, the genomic Sir2p hypothesis is appealing and substantive. In this theory, NAD acts as a catalyst for the enzymatic protein Sir2p to remove acetyl groups (a process called deacetylation) from a substance, chromatin, that normally plays a significant role in expressing the genetic messages of DNA.

Chromatin is like a loosely fitting jacket on the chromosomes; once deacetylated, it "shrink-wraps" itself to the DNA, thereby blocking or silencing genetic messages that are, in effect, cellular death notices. For reasons not altogether clear, these messages constitute the core programming of a cellular "aging clock," which, if not halted, shuts down cells one by one, eventually leading to a cascade that becomes an avalanche. Death follows.

SIR2P AND NAD MAY SLOW THE AGING CLOCK
Normally, most NAD is used for cellular metabolism, such as in the Krebs cycle, and little remains for other purposes. But when caloric consumption is restricted (by about 30% in most studies),³ the reduced metabolism in the energy-deprived cells may direct part of its action toward increasing the availability of NAD, which, in turn, upregulates the deacetylation of chromatin by the Sir2 proteins, thereby causing the gene silencing.⁴ In caloric restriction, gene silencing may slow age-related processes, such as genome instability and gene expression not appropriate for the health of the organism.

Thus, Sir2p amply in league with NAD may slow the cellular aging clock. However, because the process of cellular decline caused by ERCs may start at an earlier age and may not be reversible, it is not clear that the discipline of caloric restriction is as valuable when started later in life.⁵ Only one study has shown this to be true. Furthermore, caloric restriction is not easy: it is a Spartan ordeal of deprivation that involves forgoing one of the pinnacles of life: great food.

CORROBORATION FOR THE GREAT DISCOVERY
The great discovery of Leonard Guarente and colleagues of the essentiality of NAD in the mechanism of Sir2p - the excitement of their discovery has ricocheted through every molecular genetics, biochemistry, microbiology, and cell biology lab on the planet - has now been substantiated by scientists from the State University of New York at Stony Brook. A team led by Rolf Sternglanz has found that Sir2p - a protein found in all biological kingdoms - catalyzes an NAD-nicotinamide exchange reaction that results in the deacetylation of the chromatin, and gene silencing.⁶ The NAD requirement of Sir2p for the deacetylation reaction suggests that this protein may be a sensor of the energy or oxidation state of cells.

THE BREAK AT THE RING
The first researchers to suggest the involvement of NAD in the Sir2p process were Roy Frye at the University of Pittsburgh⁷ and Danesh Moazed of Harvard Medical School.⁸ They proposed that Sir2p might operate to silence genes, not by removing acetyl groups from the chromatin but by attaching a chemical group called ADP-ribose. NAD, they conjectured, played a weak and subservient role. Sternglanz and associates disagreed. They believe that NAD is essential and that the Sir2p enzyme cleaves NAD into its two principal component parts, the ring-shaped nicotinamide molecule and ADP-ribose (which contains four rings).

In The Lord of the Rings, when the ring is set free from the bearer - by being tossed through the Cracks of Doom to the point of its creation - darkness retreats, and light returns to Middle Earth. While this is just a fantasy - albeit one of great metaphorical power, bestowed with the accumulated wisdom of epic tales and ring-quest mythology - ring structures are generally stable units of molecules. The break that occurs when Sir2p cleaves NAD, governed in part by NAD's catalytic activity, ultimately results in keeping the cell's lights on, and the darkness of cell death retreats.

THAT MIGHT BE DEACETYLATION
When Shin-ichiro Imai, a postdoc in Guarente's lab, mixed Sir2p, NAD, and part of a histone (a small protein commonly found in association with DNA in chromatin) in a test tube to see if Sir2p added ADP-ribose to the protein tails, he was shaken by the results.⁹ The histone molecules did not get heavier, as they would if weighed down by an extra ADP-ribose group. Instead, many of them got lighter by exactly 42 atomic masses. Guarente was reported to shout, "That might be deacetylation!" He knew that 42 atomic masses was exactly the loss that would occur if one of the acetyl groups were removed. Instead of simply supplying the ADP-ribose groups, NAD was acting as a catalyst: facilitating, without directly participating in, the reaction by which Sir2p removes acetyl groups from chromatin. NAD had never before been known to behave as a catalyst.

As they discovered, Sir2p plays two roles: it adds ADP-ribose groups to molecules, and it removes acetyl groups. What excites Guarente most is the possibility that Sir2p and NAD are the answer to the riddle of how semistarvation prolongs life. "Clearly, there's some link between energy, Sir2p, and aging," he says.

Guarente's hypothesis: the quantity of Sir2p affects life expectancy in yeast, rodents, primates, and probably humans.¹⁰ But Sir2p needs NAD, without which it cannot operate. In normal metabolism, not restricted by reduced caloric consumption, NAD is employed mainly for the production of cellular energy. But when calories are restricted and metabolism slows, the amount of NAD available for the gene-silencing activity of Sir2p should increase, thus extending the lifespan of the organism (see Figure 4).

Figure 4.
The possible allocation of NAD resources. With a high-calorie diet, glycolysis
 commandeers most of the available NAD, leaving relatively little to assist the
Sir2 protein in its gene-silencing task. With caloric restriction, more NAD
is available for gene silencing.

OTHER RESEARCHERS CHIME IN
C. elegans (the longevity flatworm) researcher Cynthia Kenyon agrees. "What Lenny's done is raise a very, very nice possibility. You could imagine silencing having a specific effect on genes related to aging." For Kenyon, the best thing about Guarente's theory is that it provides a new way to think about aging that is highly testable. Guarente's lab is already studying worms and mice engineered to make too much or too little Sir2 protein.

Should an abundance of Sir2p be found to extend the lifespan of these animals, as it does in yeast, it will be more possible to confirm whether it produces the same kinds of changes as caloric restriction. Already there is a positive rumble about the early findings. Curiously, Kenyon's work has shown that one of the longevity genes found in C. elegans, called the daf2 gene, modulates the activity of an insulin/IGF-1 (insulin-like growth factor) pathway. Glucose appears again.

NICOTINAMIDE SUPPLEMENTATION MAY BE THE ANSWER
Together these studies go a long way toward revealing whether Sir2p is the missing link in the quest to understand the antiaging benefits of caloric restriction. Fortunately, there is a way to increase NAD in the body, and that is via a direct precursor, the nutrient niacin (vitamin B3), in the form of either nicotinic acid or nicotinamide. Both of these have a long history of uses as nutrient supplements. The result of one study showed that, although both of them produce NAD, the rate of synthesis from nicotinamide is twice as great as that from nicotinic acid under physiological conditions, with the conversion accelerated significantly by inorganic phosphate, but only for nicotinamide.¹¹

NEUROPROTECTION
Other studies have shown that nicotinamide can help prevent DNA fragmentation. Independently of NAD's role, nicotinamide can directly help protect mice from the oxidative stress associated with various neurodegenerative, age-related diseases. In one study, it was found to produce neuroprotective effects via increased NAD levels, which rose by 50% in the mouse brain.¹² It is believed that nicotinamide helps prevent the critical depletion of NAD, thus enabling it to repair DNA.

James David Adams, Ph.D., Associate Professor of Molecular Pharmacology and Toxicology, University of California, San Francisco, has introduced the use of high-dose nicotinamide to minimize DNA damage and prevent necrosis (cell death) and apoptosis (cell suicide) in the brain.¹³ He recognizes that nicotinamide is another form of vitamin B3 and is a precursor to NAD in the brain, heart, and other organs. According to Dr. Adams, "Without nicotinamide treatment, the brain can become depleted of NAD, which leads to ATP depletion and cell death from necrosis or apoptosis. Nicotinamide is providing new perspectives on the prevention and treatment of neurodegeneration."

Apoptosis is a characteristic form of cell death (often called "cell suicide") that has been implicated in nerve degeneration. In another study, apoptosis and DNA fragmentation were induced in mice by a potent free-radical neurotoxin that damages dopaminergic (motor-mechanism) neurons in the substantia nigra of the midbrain.¹⁴ Nicotinamide was able to block the induced apoptosis and, as well, quench some of the free radicals formed by xanthine oxidase.

Nicotinamide has also been found to help protect against DNA damage induced by radiation, especially when the cells to which it was directed were repair-deficient, but not so deficient as to be defunct.¹⁵

YOUTH RESTORATION
When old mice were given nicotinamide, their susceptibility to a strong chemical oxidant that causes DNA damage in most regions of the brain was reduced to that of much younger mice.¹⁶ Without nicotinamide, as measured by cell suicide (apoptosis), older mice (24 months) were found to have significantly more DNA damage than younger ones (8 months). This is the same type of DNA-implicated nerve degeneration observed in both Alzheimer's and Parkinson's disease patients. Nicotinamide was able to prevent DNA fragmentation damage when it was coadministered with the chemical toxin in older mice.

When the white blood cells known as lymphocytes, which help fight infection and disease, are examined from old mice, they are seen to possess lower levels of DNA repair activity compared to lymphocytes from young mice.¹⁷ But when these cells were taken from mice of both age groups and treated with nicotinamide, the old mice recovered their DNA repair mechanisms (against damage by UV radiation) to twice as great an extent as did the young mice. However, when chemicals that inhibited the production of NAD from nicotinamide were present, the DNA repair activity was limited - an effect that could be overcome with a sufficient excess of supplemental nicotinamide.

NICOTINAMIDE FOR DIABETES AND OSTEOARTHRITIS
Several human trials are currently underway to find out whether nicotinamide can prevent insulin-dependent diabetes in predisposed subjects.¹⁸ The impetus for these trials was a study showing that nicotinamide could do this in mice.¹⁹ Fifty-six recently diagnosed, insulin-dependent diabetic patients were given nicotinamide daily at 25 mg per kg of body weight (2.0 g for a 175-lb individual) or a placebo, for 12 months. The results showed that nicotinamide can be added to insulin in these patients to help prevent insulin-producing beta-cell destruction.²⁰ Since the byproducts of diabetes (advanced glycation end products, or AGEs; see articles in this issue) help accelerate the aging process,²¹ it might be a good idea to take nicotinamide supplements as a preventive measure.

In osteoarthritis and rheumatoid arthritis, nicotinamide has been found to be beneficial. A recent pilot study found nicotinamide able to increase joint mobility by 4.5 degrees, vs. controls.²² The study also reported improvement in joint flexibility and inflammation abatement, while allowing a reduction in standard anti-inflammatory medications.

NAD IS NOT SIGNIFICANTLY INCREASED BY NADH
Recently popularized as a supplement, the chemically reduced form of NAD, called NADH, has been shown to be of value for enhancing brain and body functions in Alzheimer's disease, Parkinson's disease, and chronic fatigue syndrome. However, when Guarente and colleagues studied NADH, they found that it did not promote a significant level of gene-silencing by Sir2p, as did NAD.²³ Apparently NADH diverts from some enzymatic step needed to amplify Sir2p's silencing effects.

DNA ENABLE YOUR FUTURE
There are exciting developments now occurring in frontier areas of science that will result, sooner or later, in major breakthroughs in genetic engineering. Viral vector gene therapy,²⁴ antisense gene manipulation,²⁵ nanomedicine,²⁶ robotics,²⁷ and other exciting developments will change everything for the better. However, many of these developments are indefinite and dependent on a great many "if's." The challenge for most of us is to stay healthy and to use our health budget wisely, so that we're present when the biomedical future arrives.

Right now, while it seems that the genomic Sir2p theory of aging, as expounded by MIT's Leonard Guarente, is on the right track, there are no significant adverse effects in supplementing with relatively large amounts of gene supplements such as nicotinamide. But because nicotinamide alone is not enough to ensure the future, even if it does everything we think it might, it is important to take a wide range of vitamins, minerals, amino acids, phytonutients, and hormones to stay healthy.

Twenty years of passionately researching the biomedical literature has helped refine our skills here at Life Enhancement, so that we have been able to stay continuously abreast of the latest literature, frequently publishing far ahead of others in the field. We have been the first to write about and promote the use of DHEA, pregnenolone, 5-HTP, vinpocetine, mastic, and gene-support supplements, among many others.

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