Thursday, September 25, 2014

Testing the Fountain of Youth in the lab

Its 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.

Weve discussed sirtuins here a number of times before – heres 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 its 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 its 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 Ill 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 its 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 – theres 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). Dont 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. Thats 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 theres still the possibility that it isnt sufficient by itself. Its possible that CR has other effects that facilitate deacetylation – CR may cause expression or activation of other enzymes that are needed. Its 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 werent sufficient, as it was pretty clear that SIRT3 (absent in the knockout mice) was necessary, at least as far as AHL is concerned. Its still possible that SIRT3 isnt necessary for anti-aging effects of CR in tissue types that werent 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. Theres 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 doesnt work if SIRT3 is absent.)

So whats the bottom line here? Its 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)

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