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)

Rapamycin and lifespan extension

Will a pill containing the immunosuppressant drug rapamycin someday extend human lifespan a few years? In spite of the hopeful research announcements that appeared a few days ago, I wouldn't recommend getting one's hopes up just yet.

This is a topic I've discussed before: Calorie restriction, TOR signaling, and aging. And for related stuff on mTOR: here.

The executive summary is that inhibition of mTOR signaling has been shown to extend lifespan in yeast, roundworms, and fruit flies. Mice can now be added to this list, in experiments that included rapamycin in their diet.

Here's the press release:

Easter Island Compound Extends Lifespan Of Old Mice: 28 To 38 Percent Longer Life (7/8/09)

On July 8, in the journal Nature, The University of Texas Health Science Center at San Antonio and two collaborating centers reported that the Easter Island compound – called "rapamycin" after the island's Polynesian name, Rapa Nui – extended the expected lifespan of middle-aged mice by 28 percent to 38 percent. In human terms, this would be greater than the predicted increase in extra years of life if cancer and heart disease were both cured and prevented.

Although rapamycin and some related compounds have been investigated as anti-cancer therapies, the hypothesized lifespan-extending benefits are thought to be related to the by now well-documented benefits of calorie restricted diets. (For very recent news on that front, see here, for example.)

Aging researchers currently acknowledge only two life-extending interventions in mammals: calorie restriction and genetic manipulation. Rapamycin appears to partially shut down the same molecular pathway as restricting food intake or reducing growth factors.

It does so through a cellular protein called mTOR (mammalian target of rapamycin), which controls many processes in cell metabolism and responses to stress.

A decade ago, Dr. [Dave] Sharp proposed to his colleagues that mTOR might be involved in calorie restriction. "It seemed like an off-the-wall idea at that time," Dr. Richardson said.

Experiments were performed in parallel at three separate research centers and consisted of feeding hundreds of mice, starting at an age of 20 months, a diet containing a special formulation of rapamycin designed to evade breakdown in the digestive system. It was found that the age at which 90% of mice had died rose from 1,078 days to 1,179 days in male mice, compared to controls, and from 1,094 days to 1,245 days in females. The total lifespan extension, on average, was therefore 9.4% in males and 13.8% in females.

Note that some accounts of the research claim lifespan extensions of 28% to 38%, but this is misleading, since those figures represent the extension of the "old age" period of mouse life beginning at 20 months. They do not mean that the mice lived up to almost 40% longer in total. (Some pretty shoddy reporting going on here....) And there was no particular evidence to indicate that extensions of such size would have occurred if the special diet began at an earlier age. However, in experiments still going on, there is evidence for some extension when addition of rapamycin to the diet begins for mice 270 days old.

Of course, even an extension of human lifespan in the 10% range – 7 or 8 years – would be quite an accomplishment, provided quality of life in the final years remained about where it is today. (Which is a big if.)

But there are various reasons to suspect that even a 10% extension in humans is rather optimistic. Some reasons:

1. Rapamycin is an immunosuppressant, currently used therapeutically to prevent organ transplant rejection. The experimental mice were maintained under conditions that carefully protected them from infection – conditions that would not be realistic for humans.
   
2. Although mice and humans are both mammals, their genetics are not all that similar. The complete sequence of the mouse genome was recently announced (see here), and it turns out that about 20% of mouse genes are different from human analogs, or not found in humans at all. (It's been 90 million years since the last common ancestor of mice and humans.)
   
3. Rapamycin is known to inhibit an important protein kinase called mTOR (mammalian target of rapamycin). mTOR plays a key role in regulating cell growth, proliferation, and survival, so it's not all that surprising that rapamycin might affect cell biology relevant to aging and longevity. This same property of rapamycin makes it interesting as an anti-cancer agent. Rapamycin and similar compounds that inhibit mTOR have in fact been found to have anti-cancer properties in animal models. Several analogs of rapamycin have been investigated as anti-cancer therapies, and one has even been approved for human use (Torisel). But even in the anti-cancer setting, mTOR inhibitors haven't yet been slam-dunk successes.
   
4. It is not clear that rapamycin in these experiments was working the same way as calorie restriction. None of the rapamycin-fed mice lost body weight, and calorie restriction usually works best when started relatively early in life.
   
5. Experimental mice that received rapamycin got a dose of 2.24 mg per kg of body weight. That's quite a lot – about 30 to 60 times (per kg) what would be given to a 60 kg human for immunosuppression.
   

The unfortunate truth is that cell signaling pathways that affect cell growth, proliferation, and survival are rather complicated, and any interventions in such pathways are very likely to not have the expected effects and/or to have various unexpected side-effects. Here's a diagram of just some of the important pathways mTOR is involved in. Imagine that were an electrical circuit and you made ad hoc changes to important components of the circuit.... Perhaps you can see how trying to affect mTOR in order either to control cancer or enhance longevity might be a dicey proposition.

In spite of all the reservations, there are still promising signs for the role of mTOR inhibition in lifespan extension. The mechanism of action need not be the same as calorie restriction, even though that hasn't been ruled out either. For example, TOR is known from yeast and nematode studies to promote protein production in ribosomes and to inhibit protein degradation via autophagy. Invertebrate studies have shown that reversal of these TOR effects can increase lifespan. And TOR signaling is also known to influence cell growth, cell-cycle progression, mitochondrial metabolism, and insulin-analog signaling.

Remember what we said about the diversity of effects of mTOR signaling? That's definitely a sword that can cut both ways – it's powerful, but hard to predict and control. We need to understand a lot more of the biological details – otherwise we're just swinging the sword in the dark.

Harrison, D., Strong, R., Sharp, Z., Nelson, J., Astle, C., Flurkey, K., Nadon, N., Wilkinson, J., Frenkel, K., Carter, C., Pahor, M., Javors, M., Fernandez, E., & Miller, R. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice Nature DOI: 10.1038/nature08221

Further reading:

Tests raise life extension hopes (7/8/09) – BBC news story

Immune drug boosts lifespan (7/8/09) – TheScientist.com

Fountain of Youth on Easter Island? (7/8/09) – ScienceNOW

Cancer Drug Delays Aging in Mice (7/8/09) – Wired.com

A pill for longer life? (7/8/09) – Nature.com

Ageing: A midlife longevity drug? (7/8/09) – Nature.com PDF

Rapamycin extends life in mice, raising hopes of life-prolonging drug for humans (7/9/09) – The Times (UK)

What Does Life-Extending Drug Mean for Humans? (7/9/09) – Time

New clues in search for elixir of youth (7/9/09) – New Scientist

Antibiotic Delayed Aging in Experiments With Mice (7/8/09) – New York Times

First Drug Shown to Extend Life Span in Mammals (7/8/09) – Technology Review

Longevity pill on the horizon? (7/10/09) – press release

Rapamycin: “An anti-aging drug today”? (3/6/07) – Ouroboros blog post

Tags: rapamycin, biogerontology, aging, longevity

Resveratrol is getting rather confusing

Here, have another glass of this great Cabernet...

But hold on about jumping to conclusions. The resveratrol story keeps getting more complicated, according to the most recent research. This is in addition to what we just discussed here.

To begin with, there is additional confirmation that resveratrol does confer health benefits – at least in mice. However – and this is a major qualification – the research did not indicate there was a general extension of longevity in the mice.

Furthermore, the way that the research was announced introduced further confusion. In one press release (from the publisher, Cell Press, of Cell Metabolism) we have:

Red wine ingredient wards off effects of age on heart, bones, eyes and muscle (7/3/08)

Large doses of a red wine ingredient can ward off many of the vagaries of aging in mice who begin taking it at midlife, according to a new report published online on July 3rd in Cell Metabolism, a Cell Press publication. Those health improvements of the chemical known as resveratrol—including cardiovascular benefits, greater motor coordination, reduced cataracts and better bone density—come without necessarily extending the animals' lifespan.

Sinclair and de Cabo's team further show evidence that resveratrol mimics the beneficial effects of eating fewer calories. In mice, they found that resveratrol induces gene activity patterns in multiple tissues that parallel those induced by dietary restriction and every-other-day feeding.

But in another press release, from NIH's National Institute on Aging, we find:

Resveratrol found to improve health, but not longevity in aging mice on standard diet (7/3/08)

Scientists have found that the compound resveratrol slows age-related deterioration and functional decline of mice on a standard diet, but does not increase longevity when started at middle age. This study, conducted and supported in part by the National Institute on Aging (NIA), part of the National Institutes of Health, is a follow-up to 2006 findings that resveratrol improves health and longevity of overweight, aged mice. The report confirms previous results suggesting the compound, found naturally in foods like grapes and nuts, may mimic, in mice, some of the effects of dietary or calorie restriction, the most effective and reproducible way found to date to alleviate age-associated disease in mammals.

The findings, published July 3, 2008, in Cell Metabolism, may increase interest in resveratrol as a possible intervention for age-related declines, said NIA scientists. The authors emphasized, however, that their findings are based on research in mice, not in humans, and have no immediate and direct application to people, whose health is influenced by a variety of factors beyond those which may be represented in the animal models.

Keep in mind, this is all about the same research. Clearly, there are some differences of spin being offered here. The second announcement seems to be closer to what was actually found, as can be seen from the abstract of the actual journal article:

Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span

A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation, and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started midlife.

(Aside: in discussions like this you will often see the terms "longevity" and "lifespan" used almost interchangably. Properly speaking, "longevity" is usually the better term, as it refers to average length of life, a statistical property, while "lifespan" refers to maximum potential length of life. For the most part, the distinction can be glossed over, though it isn't entirely unimportant.)

Confused yet? Let me try to boil this down a little. This is just my interpretation, but the conclusions I see are these:

1. Resveratrol fed to middle-aged mice can have health benefits, such as cardiovascular benefits, greater motor coordination, reduced cataracts and better bone density.
   
2. The health benefits and gene transcriptional changes resulting from resveratrol in the diet are similar to, though not quite the same as, those resulting from calorie restriction alone.
   
3. Resveratrol in the diet did not increase longevity of mice on normal diets, even though calorie restriction by itself has been shown to increase longevity (in mice), while resveratrol does increase longevity of mice on high-calorie diets.
   
4. In mice on high-calorie diets where resveratrol increased both health factors and longevity, the improvement occurred without decreasing actual body weight.
   
5. This research on mice may not be predictive of the effects that might be seen in similar experiments (which have not yet been done) on humans.
   

Putting this even more succinctly, in mice adding resveratrol to the diet slows down some undesirable side-effects of aging, but does not appear to actually increase longevity, whereas calorie restriction does slow aging and increase longevity somewhat. All bets are still off as to what effects resveratrol may have in humans.

How could it be that resveratrol had all these health benefits, but didn't increase longevity? The logical conclusion would be that resveratrol has little effect on conditions that usually cause mice to die. In particular, mice usually die of cancer, and resveratrol doesn't have much benefit in that regard, though calorie restriction does.

There might actually be good news for humans in this – if resveratrol did benefit cardiovascular health in humans, that would be great, since cardiovascular disease is the largest cause of human mortality. (Cancer's a big cause too, just not as big.)

In spite of the ambiguities, this is a very significant piece of research, in part because of the large team of experienced scientists – such as David Sinclair – who participated. Here are some additional news reports on the research:

• Resveratrol helps hearts in mice, but doesn’t reproduce all the benefits of low-calorie diets (7/3/08)
  
• Wine chemical improves health but not longevity (7/3/08)
  
• Fountain of youth? Red wine gives up secrets (7/3/08)
  

But wait. We're not done yet. There are additional complications, as some of these other reports point out. For one thing, it isn't at all clear how resveratrol is beneficial at a molecular level. (And it's just as unclear why calorie restriction is beneficial – which might be different in some ways from the reasons applicable to resveratrol.) It is known that resveratrol has antioxidant and anti-inflammatory properties. Both of those are positives.

However, resveratrol also seems to activate the sirtuin protein Sirt1. There's quite a bit of research – in model organisms like nematodes, yeast, fruit flies, and rodents – that shows sirtuins have beneficial properties of their own. In particular, sirtuins do increase longevity, as does calorie restriction, in the model organisms, even if the molecular mechanisms aren't quite the same. (This is more evidence that the beneficial effects of resveratrol are not largely due to sirtuin activation.)

Regarding Sirt1 specifically, there have been at least two other recent research results published. The results are mixed. In some ways Sirt1 is shown to be beneficial, while in at least one way, it may be harmful. To the extent that resveratrol does serve to activate Sirt1, is may have the same helpful or harmful properties.

Here's the bad news, first:

Life-extending Protein Can Also Have Damaging Effects On Brain Cells (7/1/08)

Proteins widely believed to protect against aging can actually cause oxidative damage in mammalian brain cells, according to a new report in the July Cell Metabolism, a publication of Cell Press. The findings suggest that the proteins can have both proaging and protective functions, depending on the circumstances, the researchers said.

"Sirtuins are very important proteins," said Valter Longo of the University of Southern California, Los Angeles. "Overexpression can protect in some cases, and in other cases, it may do the opposite. It has to do with the fact that they do so many things." ...

SirT1, the mammalian version of yeast Sir2, controls numerous physiological processes including glucose metabolism, DNA repair, and cell death, the researchers added. In mammalian cells, SirT1 also controls several stress-response factors.

Now, the researchers show that cultured rat neurons treated with a SirT1 inhibitor more often survived treatment with oxidative stress-inducing chemicals. They further show evidence to explain the mechanism responsible for that effect.

They also found lower oxidative stress levels in the brains of mice without SirT1. However, those SirT1 knockout mice didn't live as long as normal mice do on either a normal or a calorie-restricted diet.

In brief: lowering Sirt1 levels helps cells withstand oxidative stress, while higher levels make cells more vulnerable to oxidative stress. Nevertheless, mice without Sirt1 at all live shorter lives. Confusing, no?

This research, which was published in the same issue of Cell Metabolism as the de Cabo-Sinclair study, went on to investigate in more detail what Sirt1 inhibition was doing. Here's the research abstract to explain:

SirT1 Inhibition Reduces IGF-I/IRS-2/Ras/ERK1/2 Signaling and Protects Neurons

Sirtuins are known to protect cells and extend life span, but our previous studies indicated that S. cerevisiae Sir2 can also increase stress sensitivity and limit life-span extension. Here we provide evidence for a role of the mammalian Sir2 ortholog SirT1 in the sensitization of neurons to oxidative damage. SirT1 inhibition increased acetylation and decreased phosphorylation of IRS-2; it also reduced activation of the Ras/ERK1/2 pathway, suggesting that SirT1 may enhance IGF-I signaling in part by deacetylating IRS-2. Either the inhibition of SirT1 or of Ras/ERK1/2 was associated with resistance to oxidative damage. Markers of oxidized proteins and lipids were reduced in the brain of old SirT1-deficient mice, but the life span of the homozygote knockout mice was reduced under both normal and calorie-restricted conditions. These results are consistent with findings in S. cerevisiae and other model systems, suggesting that mammalian sirtuins can play both protective and proaging roles.

(Technical aside: Note, in particular, the conjectured effect of Sirt1 on IGF-1 signaling. Sirt1 promotes phosphorylation of IRS2, the "Insulin receptor substrate 2", which enhances IGF-1 signaling, and this makes cells more vulnerable to oxidative stress. Conversely, inhibition of Sirt1 reduces cell vulnerability. (We discussed many properties of IGF-1, including relations to calorie restriction and longevity, here.))

As you recall, Sirt1 is what's called a histone deacetylase (HDAC) enzyme. (Some discussion here.) As such, one of the main properties of Sirt1 is that it can silence a bunch of genes at the same time, by removing acetyl groups from the histones to which the genes are normally bound. Clearly, that is why Sirt1 affects many diverse processes, and why it can be risky to mess with.

That's the cautionary news on Sirt1. But again on the positive side of the ledger for Sirt1 (and hence perhaps resveratrol also), there is one more study that did not receive as much media attention. The study, was published July 3 in the Proceedings of the National Academy of Sciences. Its authors included Matthias Tschöp and Paul Pfluger.

Here's the abstract (via BioInfoBank):

Sirt1 protects against high-fat diet-induced metabolic damage

Here, we report that mice with moderate overexpression of Sirt1 under the control of its natural promoter exhibit fat mass gain similar to wild-type controls when exposed to a high-fat diet. Higher energy expenditure appears to be compensated by a parallel increase in food intake. Interestingly, transgenic Sirt1 mice under a high-fat diet show lower lipid-induced inflammation along with better glucose tolerance, and are almost entirely protected from hepatic steatosis. We present data indicating that such beneficial effects of Sirt1 are due to at least two mechanisms: induction of antioxidant proteins MnSOD and Nrf1, possibly via stimulation of PGC1-α, and lower activation of proinflammatory cytokines, such as TNF-α and IL-6, via down-modulation of NF-κB activity. Together, these results provide direct proof of the protective potential of Sirt1 against the metabolic consequences of chronic exposure to a high-fat diet.

From this news story already mentioned, here's a little more explanation:

Increasing levels of the mouse sirtuin, SirT1, prevents mice from developing heart problems and fatty livers even when they are fed high-fat diets, researchers at the University of Cincinnati College of Medicine and the Spanish National Cancer Research Center in Madrid reported June 30 in Proceedings of the National Academy of Sciences. These mice with higher levels of SirT1 eat more but also burn more calories than do mice with normal levels of the enzyme.

If you haven't had enough punishment yet, here's a more detailed report on all the research already discussed, and a bit more:

SIRT1, Resveratrol and More: Moving Closer to Anti-aging Elixir? (7/8/08)

One of the additional bits is this:

Working independently and publishing 4 June in PLoS ONE, researchers led by Tomas Prolla at the University of Wisconsin, Madison, report similar results in their microarray analysis comparing transcription profiles induced by CR and resveratrol. First author Jamie Barger and colleagues fed mice from middle age (14 months) to old age (30 months) a control diet, CR diet, or resveratrol-supplemented control diet. The researchers report a “striking transcriptional overlap” of CR and resveratrol (99.7 percent of gene expression changes correlating by direction) in heart, skeletal muscle, and brain (neocortex), and show that both regimens prevent age-related cardiac problems.

And here's the journal article, in full, being referred to:

A Low Dose of Dietary Resveratrol Partially Mimics Caloric Restriction and Retards Aging Parameters in Mice (6/4/08) - also here

Resveratrol in high doses has been shown to extend lifespan in some studies in invertebrates and to prevent early mortality in mice fed a high-fat diet. We fed mice from middle age (14-months) to old age (30-months) either a control diet, a low dose of resveratrol (4.9 mg kg⁻¹ day⁻¹), or a calorie restricted (CR) diet and examined genome-wide transcriptional profiles. We report a striking transcriptional overlap of CR and resveratrol in heart, skeletal muscle and brain. Both dietary interventions inhibit gene expression profiles associated with cardiac and skeletal muscle aging, and prevent age-related cardiac dysfunction. Dietary resveratrol also mimics the effects of CR in insulin mediated glucose uptake in muscle. Gene expression profiling suggests that both CR and resveratrol may retard some aspects of aging through alterations in chromatin structure and transcription. Resveratrol, at doses that can be readily achieved in humans, fulfills the definition of a dietary compound that mimics some aspects of CR.

If that sounds a bit familiar, it's because not only does it parallel the research reported by Sinclair and de Cabo discussed above, but in fact we're already written about it here, as it was described in this press release.

To summarize this whole thing, resveratrol has benefits for both general health and longevity. The benefits are similar to, but not quite the same as, those of either sirtuins or calorie restriction. Further, sirtuins, and hence resveratrol, may also have detrimental side effects. A lot more research, which must eventually include human studies, is needed.

There have also been a couple of other recent reports on completely different possible mechanisms to explain the benefits of calorie restriction, but we'll save those for another time.

Additional reading:

The Ongoing Saga of Sirtuins and Aging – overview in Cell Metabolism of the research by Li, et al (sub rqd)

Tags: resveratrol, sirtuins, aging, longevity, calorie restriction, biogerontology

IGF-1, calorie restriction, exercise, and longevity

Loyal readers here (both of you) may recall that back here I mentioned the hormone IGF-1 and promised to deal with it more throughly. The occasion was that IGF-1 is a growth factor, like BDNF.

Basically, a growth factor is a protein for signaling between cells. Growth factors typically bind to specific receptors on a cell's surface, in order to promote cell survival, growth, or proliferation.

The following recent news item now gives me an excuse to make good on my promise:

Hormone May Hold Key To Helping Elderly Men Live Longer (5/27/08)

Elderly men with higher activity of the hormone IGF-1--or insulin-growth factor 1--appear to have greater life expectancy and reduced cardiovascular risk, according to a new study.

IGF-1 is a hormone similar in molecular structure to insulin. It is released from the liver and plays an important role in childhood growth and continues to have anabolic effects in adults. ...

Subjects with the lowest IGF-1 function had a significantly higher mortality rate than subjects with the highest IGF-1 bioactivity. These results were especially significant in individuals who have a high risk to die from cardiovascular complications.

So, does that mean we need to find ways to increase our body's IGF-1 production in order to extend lifespan? Well, not necessarily. It's more complicated than that, as we'll see shortly.

Any hint of longevity enhancement, of course, is something worth paying attention to, but in the case of IGF-1, there's a lot more to the story. It's actually kind of a big deal for several additional reasons.

To begin with, the full name of the hormone is insulin-like growth factor. It is so-named because, as the news item mentions, its molecular structure is similar to that of insulin.

But that's just the beginning of the similarity. Both IGF-1 and insulin affect metabolism. In fact, IGF-1 can bind to the same receptor that insulin does, although a lot less strongly. That, and the not coincidental structural similarity to insulin suggest that perhaps, sometime far back in evolution, the same gene may have coded for some ancestor of both insulin and IGF-1.

If you take into account a striking fact about the IGF-1 receptor, this hypothesis of a common origin for insulin and IGF-1 becomes even more intriguing. The fact is that the (gene for the) IGF-1 receptor is a homologue of the daf-2 gene of the nematode Caenorhabditis elegans (as is the gene for the insulin receptor also). In fact, DAF-2 (the protein product of daf-2) is the only insulin-like receptor in nematodes, so biologists now regard daf-2 as the ancestor of the mammalian receptors for IGF-1 and insulin.

I first mentioned this relationship back here, and went into more detail here, in connection with understanding the effect of sirtuin proteins on aging and longevity of C. elegans.

But the "coincidences" don't stop there. The important function of a receptor is the effect it has, when activated, upon signaling downstream inside the cell. All of the receptors we're talking about here are of the sort called tyrosine kinase receptors. Let's unbundle that term. Tyrosine is one of the 20 amino acids that make up proteins. A kinase is a type of protein enzyme whose function is to attach phosphate groups to specific kinds of amino acids in other proteins. This process is called phosphorylation. When another protein of the right sort is phosphorylated, it becomes able to act as a tyrosine kinase itself, and go on to affect yet other proteins.

This whole process is called signal transduction. The process begins (in the case here) with a receptor tyrosine kinase, which is a cell surface receptor protein that is also a tyrosine kinase – for example DAF-2, and the receptors for IGF-1 and insulin. There may be a number of intermediate steps, but the eventual result is the phosphorylation of a transcription factor, which enters the cell nucleus and facilitates the transcription of certain genes in order to produce new proteins.

In C. elegans, DAF-16 is the transcription factor that is activated by signaling mediated by DAF-2. We discussed DAF-16 in the aforementioned posts here and here. DAF-16 belongs to a family of transcription factors called forkhead box proteins. We have discussed these before too, or rather the subclass called FoxO transcription factors.

We're getting pretty far into the technical weeds here, so if you want more details on this stuff, refer to the earlier posts.

To make the long story short, the effects of the external signaling hormones like insulin and IGF-1 ultimately result from proteins coded for by the genes expressed because of the appropriate transcription factors that were activated by the signaling cascade. There are probably many proteins involved, and sorting them all out, figuring out how they collectively affect longevity, is very much an ongoing project.

The story is interesting to understand because longevity is one of its main themes. In addition to the news item already mentioned, there's more recent news with the same theme. Here are summaries of some of these research announcements:

When It Comes To Living Longer, It's Better To Go Hungry Than Go Running, Mouse Study Suggests (5/14/08)

It is once again verified that a low-calorie diet can extend the lifespan of rodents. This benefit is beyond what can be achieved with a higher-calorie diet offset by exercise. However, rats that consumed the most calories, and has less longevity, also had the highest levels of IGF-1. Rats that consumed the fewest calories had the best longevity and the lowest levels of IGF-1. Exercise could only partially counteract the higher IGF-1 levels and reduced longevity of rats on a high-calorie diet. In this study, IGF-1 levels were inversely correlated with longevity. This is a "live-fast, die-young" scenario, which is especially typical of rodents, but not necessarily of humans.

More on this study: here

Shorter Women May Have Very Long Lives: Gene Mutation Found (3/4/08)

This study focused attention on the (adult) daugheters of especially long-lived Ashkenazi Jews. A control group consisted of daughters of the same age as the others, but whose families had no history of unusual longevity. The finding was that female children of long-lived individuals (aged 95-110) were on average 2.5 cm shorter than female controls. It was also found that both the centenarians and their daughters were much more likely than the controls to have mutations in the genes for their IGF-1 receptors. However, the daughters also had blood plasma levels of IGF-1 that were 35% higher than the levels in the control group. The interpretation is that the higher IGF-1 levels were due to an attempt to compensate for disruption of IGF-1 signaling due to irregularities of the receptor proteins. This would be consistent with a number of animal studies in which reduced IGF-1 signaling correlates with increased longevity.

More on this study: here, here, here

Interestingly enough, IGF-1 had already been recognized to have an effect on body size – in mice and dogs. The dog research is described here:

One gene between tiny dogs and giant ones? (10/13/06)

Nate Sutter, a geneticist at the National Human Genome Research Institute in Bethesda, Maryland, wanted to know the reason why big dogs, such as Irish wolfhounds, can grow up to 50 times larger than other members of their own species, such as chihuahuas. So he started out looking at large and small dogs of one breed — the Portuguese water dog. ...

The team found that one of the few differences in these Portuguese water dogs occurred in a gene called 'insulin-like growth factor 1', or Igf-1 .

This is one of many genes already known to influence the size of mice: when Igf-1 is knocked out, the animals grow up to be mini-mice.

(The article is subscription-only, but you can find another reference to it here.)

The researchers went on to do further analysis of the IGF-1 gene in many different dog breeds of all sizes, and also in foxes and wolves. They found that almost all of the small breeds had the same variant of the IGF-1 gene as the small Portuguese water dogs had, while almost none of the large breeds had that variant. The team concluded that the IGF-1 variant in small breeds is responsible for the difference because it reduces production of the growth factor.

This should also explain what dog people have always known – that small breed dogs generally live longer than large ones.

Here's a later report of the same research:

What Makes Little Dogs Small? Researchers Identify Gene Involved In Dog Size (4/5/07)

In their study, researchers explored the genetic basis for size variation among dogs by comparing the DNA of various small dog breeds, including Chihuahuas, Toy Fox Terriers and Pomeranians, to an array of larger dog breeds, including Irish Wolfhounds, Saint Bernards and Great Danes. Their investigation found that variation in one gene - IGF-1, which codes for a protein hormone called insulin-like growth factor 1 - is very strongly associated with small stature across all dog breeds studied.

Further reading:

Scientists Explore Queen Bee Longevity (5/8/07) – press release describing research on various factors, including IGF-1 signaling, in queen bee longevity

Mechanisms of lifespan regulation by IGF-I (2/25/08) – blog post that considers some of the paradoxical effects of IGF-1 that may be beneficial in some ways but also shorten lifespan

Not so fast, daf-2: IGF-I is all kinds of good for you (1/23/08) – another blog post on the paradoxical effects of IGF-1

IGF-1 attenuates cardiac aging (11/15/06) – blog post about research on cardioprotective properties of IGF-1

It’s not easy being wee: Does IGF-1 deficiency slow down the brain? (8/30/06) – one more blog post on paradoxical effects of IGF-1

A Single IGF1 Allele Is a Major Determinant of Small Size in Dogs – 4/6/07 research article in Science (sub. rqd.)

Tags: IGF-1, calorie restriction, longevity, aging

Adiponectin, longevity, and cancer

Adiponectin is a hormone that is made exclusively in adipose (fat) tissue and secreted into the blood stream. It modulates a number of metabolic processes, such as glucose regulation and production of energy from fatty acids.

We had a long note on adiponectin last September (here), which has turned out to be very popular. That article summarized a number of research results concerning adiponectin that have appeared in the last few years. Undoubtedly, much of the interest in adiponectin is a result of its relevance to such things as weight control, diabetes, inflammation, cardiovascular disease, and kidney disease.

Some research that was reported in April had more to say about the relation to kidney disease:

Fat-cell Hormone Linked To Kidney Disease (4/22/08)

Reduced levels of a hormone produced by fat cells and linked to the development of insulin resistance may also be related to a higher risk of kidney disease, according to a study led by researchers at the University of California, San Diego School of Medicine and Thomas Jefferson University. ...

The new findings show that the hormone, adiponectin, produced by fat cells, circulates in the blood and acts to both suppress inflammation -- known to be a contributor to diabetes and cardiovascular disease -- and to reduce protein in the urine.

"A deficiency in adiponectin could be the major reason why obese patients develop the initial signs of kidney disease," said principal investigator Kumar Sharma.

The research showed that adiponectin promotes proper function of kidney cells called podocytes:

A network of fine capillaries in the kidney acts as a filter to prevent proteins in the blood from being secreted into the urine. This filter is made up of three components, one of which -- the podocyte cell -- serves to regulate albuminuria.

"We discovered that the hormone adiponectin, produced by fat cells, is directly linked to the healthy function of podocytes," said Sharma.

While that's interesting, it's not clear that this activity has much to do with adiponectin's effect on metabolism through favoring the use of fats as a source of energy instead of glucose. This may be a case where an important hormone really does have unrelated effects on different physiological systems.

Earlier research on adiponectin suggested that it served as a signal of low levels of available food calories, and hence caused the body to favor metabolism of stored fat as an energy source. This could well be related to the known effects of calorie restriction on longevity. Indeed, some research from last November suggested that longevity is promoted because metabolism of fat generates a lower level of reactive oxygen species than does metabolism of glucose:

Fat Hormone May Contribute To Longevity (11/21/07)

Using a mouse model of longevity, Terry Combs and colleagues report that changes in metabolism can indeed increase longevity. They demonstrated that long-lived Snell dwarf mice burn less glucose and more fatty acids during periods of fasting, and as a result produce fewer free radicals.

The key to this switch may be adiponectin, a hormone produced by fat cells that helps lower glucose production and stimulates cells to use fat for energy instead. The researchers found that Snell mice had three times as much adiponectin in their blood as control mice; Snell mice also had fewer triglycerides in their cells, indicative of higher fat metabolism.

The benefit of burning fats instead of glucose for energy is that it produces fewer oxygen radicals which can damage cells and exacerbate the effects of aging. Confirming this, Combs and colleagues found far less free radical damage.

Given that reactive oxygen species are also linked to increased inflammatory response and DNA damage, and that both of these effects are linked to cancer, it's not too surprising to find that variations in the gene for adiponectin may affect cancer risk:

Gene Variations May Predict Risk Of Breast Cancer In Women (5/2/08)

According to a recent study, led by Virginia Kaklamani, MD, an oncologist at Northwestern Memorial Hospital and assistant professor of medicine, Northwestern University Feinberg School of Medicine, variations of the adiponectin gene, which regulates a number of metabolic processes, may increase a woman’s risk of developing breast cancer. ...

Dr. Kaklamani’s research, which is published in the May 1 issue of Cancer Research, suggests some women are born with different characteristics in the adiponectin gene which can alter its function and increase the risk of breast cancer. This finding, coupled with previous studies that have found a correlation between low levels of adiponectin in the body and cancer risk, suggest adiponectin may be the third gene linked to breast cancer among women with no previous family history of breast cancer. If confirmed through additional studies, adiponectin could be used along with TGF-beta and CHEK2, genes that have already been linked to breast cancer, to create a genetic testing model that will allow clinicians to more accurately predict breast cancer risk.

Further reading:

Happy fat: Calorie restriction modulates adipocyte gene expression – 7/17/07 blog article that discusses research relating calorie restriction to adiponectin

Adipogenic signaling in rat white adipose tissue: Modulation by aging and calorie restriction – abstract of the research discussed in the preceding item.

Tags: adiponectin, longevity, cancer, reactive oxygen species

Calorie restriction, TOR signaling, and aging

Now that I've given some pointers to information about how TOR signaling is involved with metabolism (see here), it seems like an opportune time to mention a recent research announcement in this general area.

How Dietary Restriction Slows Down Aging (4/17/08)

University of Washington scientists have uncovered details about the mechanisms through which dietary restriction slows the aging process. Working in yeast cells, the researchers have linked ribosomes, the protein-making factories in living cells, and Gcn4, a specialized protein that aids in the expression of genetic information, to the pathways related to dietary response and aging.

Here's the key background:

Previous research has shown that the lifespan-extending properties of dietary restriction are mediated in part by reduced signaling through TOR, an enzyme involved in many vital operations in a cell. When an organism has less TOR signaling in response to dietary restriction, one side effect is that the organism also decreases the rate at which it makes new proteins, a process called translation.

The researchers investigated various strains of yeast cells that had low rates of protein production, but increased lifespan. They found that a common characteristic of such cells was mutations to one part of the cell's ribosomes, the complex of RNA and certain proteins which manufactures all new proteins in the cell. The result of these ribosome changes was a decrease in the production of most proteins, except for one, called Gcn4, a transcription factor, whose production increased. The effect seems to depend on the same pathway affected by reduced TOR signaling. Gcn4 is associated with control of amino acid synthesis, and is activated when a cell is starved for amino acids.

To make the link between Gcn4 and longevity, the scientists then asked whether preventing the increase of Gcn4 would block life span extension. In every case, cells lacking Gcn4 did not respond as strongly as Gcn4-positive cells.

"The increased production of Gcn4 in long-lived yeast strains, combined with the requirement of Gcn4 for full life-span extension, makes a compelling case for Gcn4 as an important downstream factor in this longevity pathway," Kaeberlein said.

One might speculate that increased Gcn4 production somehow helps the cell cope with lack of nutrients, and one effect is that the cell takes steps to conserve its resources and slow the rate of aging.

Since reduction of TOR signaling is one way to bring about this effect, TOR inhibitors might help slow aging and increase lifespan, at least in yeast. However, since TOR affects so many other cell functions, the chance for harmful side effects of reduced TOR signaling is high.

"The role of TOR and translation in aging is known to be conserved across many different species, so it's plausible that this function of Gcn4 is conserved as well," Kennedy said. Future research will be aimed at testing this hypothesis.

"Clearly TOR signaling is one component, and perhaps the major component, of the beneficial health effects associated with dietary restriction," said Kaeberlein. "The difficulty with TOR as a therapeutic target, however, is the potential for negative side effects. As we learn more of the mechanistic details behind how TOR regulates aging, we will hopefully be able to identify even better targets for treating age-associated diseases in people."

Tags: calorie restriction, TOR signaling

Sirtuin news

Back in November we had a series of posts about sirtuin proteins. This included an overview, with a particular focus on the relevance to calorie restriction and longevity, especially in light of recent research announcements at the time. This was followed by a couple of posts (here, here) on background history.

Now is a good time to return to that thread and continue the discussion of sirtuins, because of additional related research announcements, including especially this:

Sirtris Announces Positive Results with Proprietary Version of Resveratrol, SRT501, in a Phase 1b Type 2 Diabetes Clinical Study (1/7/08)

Sirtris Pharmaceuticals, Inc. ... announced today that the Company's first product to enter the clinic, SRT501, was found to be safe and well-tolerated, and was found to significantly lower glucose in an oral glucose tolerance test conducted as part of a 28 day Phase 1b clinical study in patients with Type 2 Diabetes.

This 28-day Phase 1b study was designed to assess the safety, tolerability and pharmacokinetics of once-daily, orally administered doses of either 2.5 g or 5 g of SRT501 in patients with Type 2 Diabetes who were naive to other diabetes drug treatments. Both doses of SRT501 were found to be safe and well-tolerated, and pharmacokinetics, a measure of drug levels in the blood, were identical at days one and 28, suggesting no drug accumulation. There were no serious adverse events and no dose-related adverse events. Importantly, SRT501 showed a statistically significant improvement in an oral glucose tolerance test on day 28 at two hours and a trend towards lower fasting plasma glucose levels.

SRT501 is also being tested in patients with Type 2 Diabetes in a Phase 1b BID (twice daily administration) study and in a Phase 2a study in combination with metformin, the current first-line therapy for Type 2 Diabetes. SIRT1 is the founding member of the human sirtuin family of enzymes which control the aging process. Specifically, SRT501 acts by increasing mitochondrial activity and therefore is targeted to address metabolic diseases, such as Type 2 Diabetes.

"This is the first time that a small molecule targeting sirtuins, the genes which control the aging process, has shown efficacy in a disease of aging," said Peter Elliott, Ph.D., Senior Vice President of Development at Sirtris.

OK, this is obviously a self-promotional PR piece from the drug developer. In particular, sirtuins aren't "the genes which control the aging process", merely some of them. However, if the claims hold up under further testing, especially the one stated in the last paragraph, this is an important validation of much prior research into the effect of sirtuins on longevity as a result of action in various cellular pathways. Our previous discussions reviewed some of this research conducted on model organisms like yeast and the nematode C. elegans.

More information: Resveratrol-like drug works in humans-Sirtris (1/7/08), Sirtris Anti-Aging Drug Generates Buzz, But May Already Be Old News (1/8/08)

This drug, SRT501, has been in human clinical trials for about a year and a half already. The initial trial (called "Phase 1") involved 85 healthy volunteers and began in June 2006 (see here). Results from that trial were reported in October 2006 (see here) and demonstrated that the drug was reasonably safe and well-tolerated.

SRT501 is a small molecule drug that achieves its effects by activating the mammalian SIRT1 NAD-dependent deacetylase enzyme, which has been investigated extensively for a decade (as discussed here). The drug is essentially just a proprietary formulation of resveratrol, the well-known component of red wine that has been shown to have lifespan-extending and anti-diabetes properties in several model organisms. (See here for an extended discussion, including reports of important research announced in late 2006.) SRT501, however, is a much more practical way to take advantage of resveratrol, compared to consumption of red wine, where hundreds or thousands of bottles of wine would be needed to achieve the same effect.

Perhaps the most important result shown by this newly reported result is that SRT501 actually seems to provide measurable beneficial effects of improved glucose tolerance and reduced blood glucose levels for humans with diabetes.

SIRT1 activators which are apparently much more powerful than SRT501 are under active investigation at Sirtris and in the laboratory of Sirtris co-founder David Sinclair. This has been documented in research that was published last November:

Sirtris unveils promising, novel SIRT1 activators for treating diseases of aging (11/28/07)

In November 2006, Sirtris scientists and Sirtris co-founder, Prof. David Sinclair from Harvard Medical School, published consecutive papers in the journals Cell and Nature showing that resveratrol, a SIRT1 activator found in red wine, could reduce the impact of a high fat diet, increase stamina two fold and significantly extend lifespan of mice. Unfortunately, it was estimated that a person would need to drink 1000 bottles of red wine to obtain an equivalent dose of resveratrol. Now, scientists at Sirtris have developed SIRT1 activating molecules that are chemically distinct from resveratrol and are 1000 times more potent.

"The new drug candidates represent a significant milestone because they are the first molecules that have been designed to act on genes that control the aging process. For this reason, we feel they have considerable potential to treat diseases of aging such as Type 2 Diabetes," said Christoph Westphal, M.D., Ph.D., Chief Executive Officer and Vice Chair of Sirtris Pharmaceuticals. "The breakthrough in potency we have achieved with the novel chemical entities (NCEs) means that we can obtain the health benefits of resveratrol with a considerably lower dose."

Here's a useful professional assessment of these results: Sirtuin activators as anti-diabetes drugs, and beyond (11/29/07) More: Sirtris Drug May Slow Aging, Create 'Armstrong' Cells (11/28/07)

Additional information:

Gene Believed To Promote Long Life Linked To Cholesterol Flushing (10/12/07)

Research conducted in part by sirtuin-research pioneer Leonard Guarente has established one mechanism through which SIRT1 provides health and longevity benefits. The mechanism promotes flushing harmful buildups of cholesterol in macrophage cells of the immune systems of mice. This mechanism could explain part of the health benefits of SIRT1-activators such as resveratrol and calorie restriction.

Red Wine Ingredient -- Resveratrol -- Fights Diabetes In Mice (10/4/07)

Chinese researchers have reported that relatively low doses of resveratrol can improve insulin sensitivity in mouse cells, and they believe this effect is due to SIRT1 activation by resveratrol. Additionally, the researchers found that SIRT1 levels are reduced in insulin-resistant cells, and that increased SIRT1 activity improved insulin sensitivity

Sirtris Pharmaceuticals – Treating Disease by Modulating Sirtuins

This is a brief overview of Sirtris Pharmaceuticals drug development focus.

Tags: resveratrol, sirtuins, aging, longevity, calorie restriction, SIRT1, Sirtris, SRT501, diabetes

FoxO transcription factors

Transcription factors are proteins that help regulate genes. This regulation may involve either enabling the expression of a gene or preventing expression. In the first case, the transcription factor is an "activator", and in the second case a "repressor".

Transcription factors perform their function by binding to a particular portion of DNA that is specific to a given gene. When bound to the appropriate DNA segment, a transcription factor affects gene expression by either facilitating (activator) or inhibiting (repressor) the operation of RNA polymerase in transcribing the affected gene into messenger RNA. Usually more than one transcription factor must be present to affect gene transcription, and additional proteins (called "cofactors") may also be required.

To make things even more interesting, transcription factors usually affect multiple genes, which may be otherwise unrelated to each other.

A particularly important family of related transcriptions factors comprises what are called "forkhead box" proteins, or Fox proteins, for short. (The name refers to a sequence of 80 to 100 amino acids that are part of the protein and bind to DNA, and which was originally discovered in fruit flies (Drosophila).)

Among the genes that Fox proteins are involved with are genes related to cell growth, proliferation, differentiation, longevity, and embryonic development. So there are Fox proteins that are important for things like cancer and stem cells – and thus it's quite useful to know about them.

An important subfamily of Fox proteins are the FoxO proteins, and we'll discuss some recent examples in this note.

To begin with, perhaps the most recent example is this:

Molecular Signal That Helps Muscle Regenerate Discovered (12/19/07)

Muscle regeneration after injury is complex and requires a coordinated interplay between many different processes. Key players in regeneration are muscle stem cells, so-called satellite cells. They divide and produce many new muscle cells to fix the damage incurred by injury. A crucial regulator of muscle function and repair is a signalling molecule called calcineurin. It is activated by injury and controls the activity of other key proteins involved in differentiation and the response to damage.

It turns out that calcineurin works by inhibiting FoxO.

Using sophisticated molecular techniques, the scientists revealed that calcineurin accomplishes its effect on muscle by inhibiting another protein called FoxO. FoxO is a transcription factor, a protein that plays a crucial role in skeletal muscle atrophy through the induction of genes involved in cell cycle repression and protein degradation. Suppressing the effects of FoxO, calcineurin ensures that proliferating cells stay alive and keep dividing to produce enough cells to repair muscle damage.

In this case, the normal function of FoxO is to inhibit cell proliferation (as a check on cancer), but this needs to be bypassed (temporarily) to enable muscle regeneration.

This result follows the discovery a few months earlier of the way a specific FoxO protein (FoxO1) cooperates with another important developmental protein (Notch) to control muscle cell differentiation:

Building Muscle Requires Foxo1 (8/25/07)

The mechanisms by which Foxo proteins regulate metabolism are relatively well characterized. However, little was known about the mechanisms by which these same proteins regulate cellular differentiation.

New data generated by Domenico Accili and colleagues at Columbia University, New York, now indicates that Foxo1 cooperates with Notch to control muscle cell differentiation in vitro.

Overexpression of either a constitutively active form of Foxo1 or a constitutively active form of Notch was found to inhibit the in vitro differentiation of a mouse myoblast cell line.

Note that the preceding alludes to the involvement of FoxO proteins in regulation of metabolism. This comes about because they affect the insulin signaling pathway, and hence also glucose and lipid metabolism.

This function is what allows yet another well-known protein, mTOR, to play a role in "metabolic syndrome" – a group of disorders that includes insulin resistance, heart disease and high lipid levels. (mTOR is short for "mammalian target of rapamycin". It's a protein kinase that modifies other proteins by phosphorylation.) The same mechanism appears relevant also to the "Atkins diet" and the effects of calorie restriction.

Fly Genetics Reveal Key Workings Of Atkins Diet (8/8/06)

Using fruit flies bred with a newly created mutant form of the gene TOR (short for target of rapamycin), Oldham and his colleagues were able to determine how the TOR pathway interacted with other important regulators of insulin, glucose and lipid metabolism.

TOR is an ancient gene, found in nearly all animal and plant cells. The researchers discovered that their new mutant fly reduced TOR function, allowing them to observe what happens when TOR's influence is removed.

Reductions in TOR function lowered glucose and lipid levels in the body. They also blocked the function of another important insulin regulator, a factor called FOXO, which is known to be a critical mediator of insulin signals and therefore glucose and lipid metabolism.

As if all that weren't enough, FoxO proteins are also involved with cancer and stem cells:

Gene Knockouts Reveal FoxOs' Vital Functions In Cancer Defense, Health Of Stem Cells (1/25/07)

In an elegant, multiple-gene knockout experiment, a team of Boston scientists has discovered that a trio of molecules, called FoxOs, are fundamentally critical in preventing some cancers, maintaining blood vessel stability, and in keeping blood-forming stem cells healthy. ...

The researchers at Brigham and Women's found that mice engineered to lack genes for the FoxO1, FoxO3, and FoxO4 molecules had serious blood abnormalities. Without the FoxO gene-regulating molecules, the rodents' blood stem cells -- master cells that give birth to working blood cells while also renewing themselves -- divided too fast and "burned out." ...

In the companion paper, lead author Ji-Hye Paik, PhD, of Dana-Farber and colleagues from the DePinho lab report that the three FoxO molecules, known as transcription factors, normally function as tumor suppressors that override maverick cells threatening to grow too fast and form tumors. When FoxOs are eliminated, it may allow cancer to develop.

And even that's not the end of it. FoxO proteins are also involved in the increased levels of inflammation often associated with the aging process. (This phenomenon has been tagged with the neologism "inflammaging".) It has been hypothesized that inflammaging results from the effect of phosphorylated FoxO on another notorious transcription factor, NF-κB (which is heavily involved in inflammation). Some of the effects of calorie restriction may also be due to FOXO phosphorylation. Reference: Restricting inflammaging (11/12/07)

FoxO is also regulated (as is P53) by SIRT1 – so this is yet another relationship to calorie restriction. Reference: Unlocking the Secrets of Longevity Genes

Additional references (for the seriously interested):

An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C. elegans.

Ageing: When Less Is More

Tags: Fox proteins, transcription factor, longevity, aging, calorie restriction

P53, a versatile gene

P53 is well-known for its role in regulating the cell cycle so as to suspend the cycle or even lead to cell death via apoptosis in case damage to a cell's DNA is detected. This function is especially important in forestalling cancer.

And as we noted here, p53 is also involved with skin tanning.

But that's not all p53 is good for. It also plays a role in fertility, which has recently been reported by one of the co-discoverers (Arnold Levine) of p53:

Cancer Fighter May Be Fertility Helper

A protein known primarily for its role in fighting cancer also helps embryos implant in the womb, according to a study in mice. The find may explain why some women have difficulty becoming pregnant.

More information: here, here

But the list of p53's goodness doesn't stop there. It also slows aging, apart from deterring cancer, but via the same mechanism:

Anti-cancer gene p53 doubles up as anti-ageing agent

The latest research suggests that one of the genes that protects us from cancer may also help delay the ageing process.

A new study has found that a particular gene, p53 which has been previously linked to premature ageing, along with one of its cellular regulators, called Arf, may boost the body's antioxidant activity to keep cells younger longer and thereby slow down the aging process.

The regulatory chemical Arf, lets p53 know that a particular cell is in trouble and marked for elimination.

More information: here, here, here, here

But, surprisingly, at least in fruit flies, reducing p53 activity may also increase lifespan, and apparently in the same way that calorie restriction does:

Key To Longer Life (in Flies) Lies In Just 14 Brain Cells

Two years ago, Brown University researchers discovered something startling: Decrease the activity of the cancer-suppressing protein p53 and you can make fruit flies live significantly longer.

Now the same team reports an intriguing follow-up finding. The p53 protein, they found, may work its lifespan-extending magic in only 14 insulin-producing cells in the fly brain.

How was this connected with calorie restriction? Simply by noting that calorie restriction in fruit flies didn't increase longevity when p53 activity was suppressed in only 14 insulin-producing cells of the flies' brains:

Studies have shown that low-calorie diets can significantly increase the lifespan of flies, worms, mice and rats. The phenomenon is of intense interest to researchers who study aging. They want to know if caloric restriction works in people and if drugs could be made to mimic its effects.

So researchers restricted the diets of the flies and ran the same experiments. The calorie-restricted flies didn't live any longer when p53 was reduced in the insulin-producing cells. This evidence supports the notion that p53 reduction is one of the direct effects of caloric restriction.

Even more intriguing, Helfand said, is the fact that the 14 insulin-producing cells that seem to be critical for lifespan extension are the equivalent of beta cells in the human pancreas. Beta cells make and release insulin, the hormone that controls the level of glucose in the blood. The research team found that when p53 activity drops, so does insulin-responsive activity in the fat body, the major metabolic organ in the fruit fly.

The involvement of insulin in this effect is especially interesting, as insulin signaling has also been found to be involved in the mechanism by which sirtuin proteins extend longevity in nematodes and fruit flies (and perhaps other organisms).

One wonders just how p53 came to play such a prominent role in cellular processes. Some researchers think they have found the answer – endogenous retroviruses that have actually proven beneficial to the host genome:

Ancient Retroviruses Spurred Evolution Of Gene Regulatory Networks In Humans And Other Primates

Scientists have long wondered how a master regulator such as p53 gained the ability to turn on and off a broad range of other genes related to cell division, DNA repair, and programmed cell death. How did p53 build its complex and powerful empire, so to speak?

Using the tools of computational genomics, the UCSC team gathered compelling evidence that retroviruses helped out. ERVs jumped into new positions throughout the human genome and spread numerous copies of repetitive DNA sequences that allowed p53 to regulate many other genes, the team contends.

"This would have provided a mechanism to quickly establish a gene regulatory network in a very short evolutionary time frame," said Ting Wang, a post-doctoral researcher at UCSC and lead author of the paper.

It's hard to avoid a suspicion that there's a lot to the story of p53 left to be discovered.

Tags: p53, aging, longevity, calorie restriction, insulin signaling