Testing the Fountain of Youth in the lab

It's been more than 10 years since it was noticed that certain enzymes – the sirtuins – had life-extending properties in organisms like yeast, and later nematodes, fruit flies, and mice. The excitement spread to other compounds, such as resveratrol, that seemed to activate or assist sirtuins. Hopes were high that such things might offer the known longevity benefits of calorie restriction in a pill form. Ever since then the gold rush has been on to figure out how these things work – and if possible, to be the first to market with the Fountain of Youth in a bottle.

We've discussed sirtuins here a number of times before – here's a list of some of those discussions. If you need to brush up on the background, those would be good places to start.

The initial sirtuin that seemed to be most important for the longevity of yeast was SIR2. The gene for SIR2 is highly conserved in evolution – so it's probably kind of important. Homologs of SIR2 have been found in many sorts of higher organisms (nematodes, fruit flies, etc.). In mammals, including humans, there is a whole family of sirtuins, having at least 7 members, named SIRTx for x=1 to 7. ("SIRT" and "sirtuin" refer to SIR-two, where SIR was an acronym for "silent information regulator".)

SIR2 is primarily a histone deacetylase (HDAC), that is, an enzyme that removes acetyl groups from histone proteins (and often other types of proteins as well). Histones are the building block proteins that make up nucleosomes, around which DNA is spooled in chromosomes. Normally, DNA is tightly bound to the histones, which prevents the genes in the tightly bound portion of DNA from being transcribed into RNA in order to make proteins. In other words, the genes bound to a histone are effectively silenced. In order for a gene to be expressed, the histone closest to the portion of DNA containing the gene has to have an acetyl group attached at an appropriate location. Enzymes ("acetyltransferases") attach acetyl groups (in the process called acetylation) to histones in order to allow gene expression. Consequently, deacetylase enzymes, such as several sirtuins, are able to silence genes by removing acetyl groups from histones.

SIRT1 is the most intensively studied mammalian sirtuin. Like SIR2, it is primarily a histone deacetylase that is active in a cell nucleus to silence a wide variety of genes. Since SIRT1 can silence a large number of genes, it affects many cellular processes. However, there is one additional complication. SIR2 and SIRT1 only have their deacetylation ability in the presence of a small molecule called NAD: nicotinamide adenine dinucleotide, and only when NAD has a net positive charge, due to the loss of one electron during the process of metabolism in which cells generate needed energy. NAD⁺ denotes this form of NAD. (The neutral form of NAD is denoted by NADH.) Because of the role of NAD⁺, SIR2 is said to be a "NAD⁺-dependent" histone deacetylase.

All this is important, because research over the past 10+ years has shown that the lifespan-extending properties of calorie restriction, especially in simple organisms like yeast and nematodes, seem to be related, at least sometimes, with the deacetylation properties of SIR2 in the presence of NAD⁺. When an organism is in a calorie restricted environment, metabolism slows down, and less NAD⁺ gets used up. As a result, there is more NAD⁺ around. So SIR2 is more effective. So genes are silenced that would otherwise be expressed. Silencing these genes seem to help an organism live longer when nourishment is not ample – so that it can survive until the buffet table is restocked.

In an organism on a normal (not calorie restricted) diet, up-regulating SIR2 or otherwise enhancing its gene-silencing abilities seems to compensate for decreased amounts of NAD⁺, and thereby achieves for the organism some of the anti-aging benefits of a calorie-restricted diet without having to go hungry.

The problem is that the expression of so many different genes can be affected by SIR2 deacetylation that it's difficult to identify which genes among these are actually useful for promoting longevity or retarding aging – especially in organisms more complex than yeast or nematodes.

Now, however, research has come out involving a much less studied mammalian sirtuin, SIRT3 – Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-Related Hearing Loss under Caloric Restriction. (I recommend viewing this link, since the illustration on the page will be helpful in understanding what follows here.) In spite of caveats I'll mention toward the end, this is a very significant and well-done piece of research.

A number of properties of SIRT3 had already been observed prior to this latest research. It is, like SIRT1, also a NAD⁺-dependent deacetylase enzyme. But unlike SIRT1, its main activity is found in cell mitochondria instead of in the nucleus. Consequently, SIRT3 deacetylates mitochondrial proteins instead of histones.

Of particular interest, this SIRT3 activity was known to be associated with calorie restriction (CR), because of overexpression in CR conditions and presumably also because of the NAD⁺-dependence. For example, studies in mice have shown that CR increases SIRT3 expression in liver mitochondria. Further, in knockout mice without SIRT3 mitochondrial fatty acid oxidation problems are found. Under CR SIRT3 is also overexpressed in mouse heart cells and may protect these cells from oxidative stress-induced cell death. (However, in this case it's possible that the effect resulted from HDAC activity in the cell nucleus.) So SIRT3 seems to be associated with anti-oxidant activity. There is, additionally, mechanistic evidence that SIRT3 inhibits mitochondria-related carcinogenesis. For instance, knockout mice without SIRT3 are susceptible to breast tumors.

The latest research presents strong evidence that under calorie restriction SIRT3 is involved in suppressing oxidative damage. The evidence is based on studies of oxidative stress-induced cochlear cell death responsible for age-related hearing loss (AHL) in mice. AHL is a pretty typical example of health problems associated with aging – one that affects humans as well as mice. The research not only shows an association between SIRT3 and protection from oxidative damage, but goes deep into the apparent mechanism involved. A variety of different in vitro and in vivo experiments with knockout mice provide the evidence.

To begin with, at the highest level, the researchers found that SIRT3 is required along with CR to inhibit age-related cochlear cell death and hearing loss. The knockout mice used in this, and other in vivo experiments, had both copies of the SIRT3 gene knocked out. The rate of progression of AHL was first measured in wild type (WT) mice as controls. CR was found to delay or mitigate AHL in the controls – but not in the knockout mice. This implies SIRT3 is necessary for CR to inhibit the progression of AHL – there's no benefit of CR for this condition without SIRT3. Further, when the cochlear cells of the experimental mice were examined, it was found that CR retarded cell death in the control animals but not in the mice without SIRT3.

So the key process to be concerned with is progressive cell death related to aging. The next experiments showed that the cell death was the result of oxidative damage. A lot of other studies have shown that CR inhibits oxidative damage to DNA, proteins, and lipids in many types of mammalian tissues. In the present research this was confirmed by examination of DNA in cochlear, brain, and liver tissues of control mice. But CR did not inhibit oxidative damage in the same tissues of the knockout mice. So SIRT3 appears to be necessary for the inhibition of oxidative damage to DNA, which presumably was responsible for accelerated cell death.

The next issue needing to be addressed is the mechanism by which CR inhibits oxidative damage. It is known that a small molecule, glutathione, is the major small molecule antioxidant in cells. Glutathione can exist in two oxidation states – reduced (GSH) or oxidized (GSSG). A high ratio of GSH to GSSG protects other molecules in the cell from oxidative damage, and GSH predominates in the healthy mitochondria of young mice. Conversely, a low ratio of GSH to GSSG is a marker for oxidative stress and/or aging. In the present research, the GSH:GSSG ratio was tested in control and knockout mice under CR conditions, at the age of 5 months. In the mitochondria of inner ear cells, as well as in brain and liver cells, it was found that the GSH:GSSG ratio increased as a result of CR in control mice, but not in knockout mice. Once again the presence of SIRT3 was shown to be necessary for an effect.

Obviously, the next thing to look at is how the GSH:GSSG ratio is controlled. The enzyme glutathione reductase (GSR) is known to be responsible for converting GSSG to GSH. So what happens is that reactive oxygen species (ROS) get soaked up in converting GSH to GSSG, and GSR reverses this to convert GSSG back to GSH.

However, in order to work GSR requires another molecule, nicotinamide adenine dinucleotide phosphate (NADPH) to do its job. NADPH is nothing but NAD, which we encountered in connection with the HDAC function of SIRT1, with a phosphate group attached. Like NAD, NADPH also exists in an oxidized form, NADPH⁺. This latter molecule predominates in mitochondria, and needs to be converted back to NADPH for use by GSR. (All this activity is really just shuffling electrons from one place to another. The pairs of molecules that mediate the activity are called "redox couples".)

So, what is it that converts NADPH⁺ to the plain old NADPH that we need? Well, that task is handled by yet another mitochondrial enzyme, isocitrate dehydrogenase 2 (Idh2). Don't despair – this is the last step! There is just one wrinkle. Idh2 is normally found in an acetylated form, in which case it is inactive. It needs to be deacetylated in order to become active and convert NADPH⁺ to NADPH. And that is precisely where the deacetylation function of SIRT3 comes into play. The researchers hypothesized that SIRT3 was needed in order to activate Idh2.

In order to test the hypothesis, they first measured acetylation of Idh2 in the control mice, with both normal and CR diets. With a normal diet, acetylation of Idh2 was substantial, but with CR there was an 8-fold decrease of acetylation. So it only remains to find the reason for that. In knockout mice, with no SIRT3, acetylation of Idh2 was "robust" with both normal and CR diets. That's a pretty good indication that SIRT3 was required for the effect. As a further indication, SIRT3 levels in the control mice were 3 times as high with a CR diet compared to a normal diet.

So SIRT3 is necessary for deacetylation of Idh2 under CR conditions, but there's still the possibility that it isn't sufficient by itself. It's possible that CR has other effects that facilitate deacetylation – CR may cause expression or activation of other enzymes that are needed. It's also possible that CR has other effects that increase NADPH independently of Idh2.

What if NADPH levels were tested directly? It was found that in the control mice NADPH did increase in all tissue types tested when a CR diet replaced a normal one, but this effect was not found in the knockout mice.

Efforts were made to use biochemical experiments (in vitro) to determine whether SIRT3 alone is responsible for deacetylating Idh2 under CR conditions. For example, another sirtuin, SIRT5, is also a deacetylase that occurs in mitochondria. Could it be helping deacetylate Idh2? The biochemical experiments indicated this was not the case.

Unsurprisingly, both normal and knockout mice were found to be leaner when fed a CR diet. Is it possible that lower body mass, especially resulting from less fat tissue, had some role in the protection from oxidative damage resulting from a CR diet? Perhaps, but other factors like that certainly weren't sufficient, as it was pretty clear that SIRT3 (absent in the knockout mice) was necessary, at least as far as AHL is concerned. It's still possible that SIRT3 isn't necessary for anti-aging effects of CR in tissue types that weren't tested (i. e. other than inner ear, brain, and liver tissue), or in mammals other than mice. The case is pretty solid for AHL in mice, but obviously there are many other age-related conditions and other species that should be investigated.

I should apologize for all the biochemical details presented here, but at least they should give you a good indication of just how complicated the effects of CR on aging and longevity can be – and probably are. There's a whole lot of work yet to be done before a reliable anti-aging pill can be developed for humans. Enthusiastic claims that this research "could lead to" therapies to slow down aging in general are basically BS. Yeah, these findings will help, but a heck of a lot more will be needed as well.

(As an example of just how badly misleading journalists who write about this stuff can be, consider this report, which begins with the claim: "In a remarkable demonstration of the ability of calorie restriction to blunt the effects of aging, scientists at the University of Wisconsin-Madison have succeeded in delaying age-related hearing loss in mice." Although the research showed that calorie restriction can do this, it did not produce any new way to do it. Instead, it shows how CR probably works by showing how CR doesn't work if SIRT3 is absent.)

So what's the bottom line here? It's pretty clear from this and many other studies that oxidative damage in cells is a cause of cell death and therefore of various health problems associated with aging. Undoubtedly there are a number of other factors that contribute to aging-related problems, such as cell death due to other causes and weakening or disregulation of the immune system. And even in the case of oxidative damage, there are many ways it can come about, and also many ways it might be inhibited. If you think of aging as a complex disease, like cancer – a point of view that has its detractors – then there are bound to be many causes and contributing factors. And also many ways to inhibit or arrest the process. The example considered here is just one of many.

Someya, S., Yu, W., Hallows, W., Xu, J., Vann, J., Leeuwenburgh, C., Tanokura, M., Denu, J., & Prolla, T. (2010). Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-Related Hearing Loss under Caloric Restriction Cell, 143 (5), 802-812 DOI: 10.1016/j.cell.2010.10.002

Further reading:

Scientists ferret out a key pathway for aging (11/18/10)

Calorie restriction delays age-related hearing loss, UW study finds (11/18/10)

Scientists ID key protein that links dietary restriction with healthy hearing, aging (12/16/10)

Calorie Restrictions Slow Aging by Enzyme Pathway (11/19/10)

Not good news for antioxidant therapies

Forget The Antioxidants? Researchers Cast Doubt On Role Of Free Radicals In Aging (2/17/09)

For more than 40 years, the prevailing explanation of why we get old has been tied to what is called oxidative stress. This theory postulates that when molecules like free radicals, oxygen ions and peroxides build up in cells, they overwhelm the cells' ability to repair the damage they cause, and the cells age.

An industry of "alternative" antioxidant therapies -- such as Vitamin E or CoQ10 supplements in megadose format -- has sprung up as the result of this theory. However, clinical trials have not shown that these treatments have statistically significant effects.

And now researchers at McGill University, in a study published in the February issue of the journal PLoS Genetics, are calling the entire oxidative stress theory into question. Their results show that some organisms actually live longer when their ability to clean themselves of this toxic molecule buildup is partially disabled. Collectively, these molecules are known as reactive oxygen species, or ROS for short.

Author summary of the open-access paper:

Deletion of the Mitochondrial Superoxide Dismutase sod-2 Extends Lifespan in Caenorhabditis elegans

In this paper, we examine the oxidative stress theory of aging using C. elegans as a model system. This theory proposes that aging results from the accumulation of molecular damage caused by reactive oxygen species (ROS). To test this theory, we examined the effect of deleting each of the five individual superoxide dismutase (SOD) genes on lifespan and sensitivity to oxidative stress. Since SOD acts to detoxify ROS, the oxidative stress theory predicts that deletion of sod genes should increase oxidative stress and decrease lifespan. However, in contrast to yeast, flies, and mice, where loss of either cytoplasmic or mitochondrial SOD results in decreased lifespan, we find that none of the sod deletion mutants in C. elegans exhibits a shortened lifespan despite increased sensitivity to oxidative stress. Surprisingly, we find that sod-2 mutant worms have extended lifespan and even worms with the primary cytoplasmic, mitochondrial, and extracellular sod genes deleted can live longer than wild-type worms. By examining genetic interactions with other genes known to extend lifespan and by comparing the phenotype of worms lacking sod-2 to that of known long-lived mitochondrial mutants such as clk-1 or isp-1, we provide evidence that the loss of sod-2 extends lifespan through alteration of mitochondrial function.

Resveratrol and cancer

Resveratrol is not a very new story these days. (For some earlier news, see here.) It is primarily known as an anti-oxidant and activator of sirtuins, but it has also been found to have specific anti-cancer properties, at least for pancreatic cancer:

Mounting Evidence Shows Red Wine Antioxidant Kills Cancer (3/25/08)

Rochester researchers showed for the first time that a natural antioxidant found in grape skins and red wine can help destroy pancreatic cancer cells by reaching to the cell's core energy source, or mitochondria, and crippling its function.

The new study also showed that when the pancreatic cancer cells were doubly assaulted -- pre-treated with the antioxidant, resveratrol, and irradiated -- the combination induced a type of cell death called apoptosis, an important goal of cancer therapy.

The fact that resveratrol has effects on the mitochondria of cancer cells is particularly interesting, since mitochondria are known to play an important role in apoptosis. One of the main ways cancer therapies such as radiation and chemotherapy kill cancer cells is by inducing apoptosis. However, advanced cancers develop ways of evading apoptosis. (See here.) If resveratrol independently interferes with the function of the mitochondria in cancer cells, that would be helpful.

In fact, the research indicated several relevant effects of resveratrol:

Laboratory experiments showed that resveratrol:

• Reduced the function of proteins in the pancreatic cancer cell membranes that are responsible for pumping chemotherapy out of the cell, making the cells chemo-sensitive.
• Triggered the production of reactive oxygen species (ROS), which are substances circulating in the human body that have been implicated in a number of diseases: when ROS is increased, cells burn out and die.
• Caused apoptosis, which is likely the result of increased ROS.
• Depolarized the mitochondrial membranes, which indicates a decrease in the cell's potential to function. Radiation alone does not injure the mitochondrial membrane as much.

More: Red wine compound may kill pancreatic cancer cells (4/14/08)

In related news, it may be worth noting that the biotech company called Sirtris, which has been developing enhanced therapeutic forms of resveratrol, recently entered into an agreement to be acquired by the pharmaceutical company GlaxoSmithKline. (See here, here, here.) This shows that the therapeutic potential of resveratrol, and other sirtuins – at least at this early stage – looks promising.


Tags: resveratrol, sirtuins, cancer

How cells can cheat death

To begin with, we have to point out that for a cell to cheat death is usually not a good thing. The reason is that cell death is usually the result of a process called apoptosis, and this process has been carefully developed (by evolution) to dispose of cells that have become "sick" because of infection (by a virus) or damage that can occur to the cell's DNA in a variety of ways. Your body needs healthy cells to function, not sick ones. Especially it does not need cells with damaged DNA, which may well become cancerous.

Apoptosis is needed in other contexts as well. In a developing embryo apoptosis is needed to remove unnecessary tissue. In addition, failure of apoptosis can lead to autoimmune diseases as well as cancer. (This is why some anti-cancer drugs are also able to treat some autoimmune diseases.)

The following research announcement, which we'll look at in more detail, has a nice capsule summary of apoptosis.

Cells Re-energize To Come Back From The Brink Of Death

Apoptosis is triggered by a variety of factors, including gene mutations that can make the cell become cancerous. During apoptosis, the membrane covering the cell's mitochondria develop holes and leak a molecule called cytochrome c, which triggers the activity of enzymes called caspases. In turn, caspases trigger a series of events that kills the cell.

To amplify a little, here are some of the conditions that can initiate a cell's apoptosis program:

• P53 protein may detect damaged DNA during the G1 phase of the cell division cycle. If it does not prove possible to repair the damaged DNA, P53 can invoke apoptosis.
  
• The cytokine TNF (tumor necrosis factor) produced by the immune system (specifically, activated macrophages) is an external signal to initiate apoptosis. As the name implies, this is another anti-cancer mechanism.
  
• Signals produced by cytotoxic T cells of the immune system can also induce apoptosis. This may occur in response to a virus-infected cell. (Much more on T cells: here.)
  

However, there is a weak spot in the apoposis process: it requires the presence of caspase enzymes. If something has blocked production of essential caspases (which some tumors are able to do), then apoptosis won't work.

Because of this, nature (i. e. evolution) has provided a backup mechanism for programmed cell death, one that does not rely on caspases. The mechanism is called, appropriately, caspase-independent cell death (CICD), and the research announcement mentioned above has this to say about it:

The process by which the membranes develop holes--mitochondrial outer membrane permeability (MOMP)--is often the "point of no return" for self-destruction, said Douglas Green, Ph.D., chair of the St. Jude Immunology department and the study's senior author. MOMP triggers apoptosis, but if apoptosis fails because there is no caspase available, the backup program called caspase-independent cell death (CICD) takes over the process.

Previous research has shown that cells that become cancerous lack caspase and other proteins needed to support apoptosis after MOMP releases cytochrome c. But this victory over death is short-lived if CICD is activated.

Unfortunately, tumors (successful ones anyway) eventually develop the ability to cheat this death program as well:

However, some cancerous cells not only dodge death from apoptosis by eliminating caspase activation, but they also foil CIDC. "Our study sought to understand how a cancer cell without caspase activation bypasses CICD as well," Green said.

The St. Jude team discovered that a cell that lacks caspase activation and cannot undergo apoptosis increases the levels of an enzyme called GAPDH in order to counteract CICD. GAPDH appears to prevent CICD by supporting the functioning of the mitochondria and triggering the activity of certain genes that prevent or repair cell damage. The findings also suggest that the increase in GAPDH provides energy to increase autophagy--the process by which a cell "chews up" debris and broken components, such as damaged mitochondria. After disposing of damaged mitochondria the cell can replace these vital components.

"We found that in the absence of caspase activation, cells that avoided CICD took about a week or so to begin multiplying again," Green said. "This might represent the time it takes for the cell to restore enough mitochondria to allow the cell to function normally."

Cancer cells are (unfortunately) amazing in their resourcefulness. Of course, this results from a kind of evolutionary process, in which cancer cells that are successful at cheating death and reproducing are those which have developed, by chance, the necessary mutations.

The role played by mitochondria and caspases in apoptosis is quite important for an understanding of both cancer and autoimmune diseases. It's worth remembering the connection, since further research will certainly tell us a lot more about these interrelated processes. Here's an example of earlier research on the subject: Proteins are Key to Cell Death in Heart Disease, Stroke and Degenerative Conditions

Tags: apoptosis, caspase, mitochondria, cancer