Posts about sirtuins

♦ Sirtuin proteins (11/16/07)

♦ The discovery of sirtuins, part 1 (11/17/07)

♦ The discovery of sirtuins, part 2 (11/20/07)

♦ Sirtuin news (1/21/08)

♦ SIRT1 and cancer (10/26/08)

♦ Testing the Fountain of Youth in the lab (2/7/11)

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)

What does marathon running do to an athlete's cells?

If you've ever taken up running as a form of exercise, or even thought about it, there's a certain paradox that may have occurred to you. The health benefits of aerobic exercise are well-documented. (See here, for example.) In particular such exercise has been shown to reduce risks of cardiovascular disease, diabetes, and some forms of cancer. Beneficial physiological effects include reduction of high blood pressure, better control of blood sugar, and reducing blood levels of low-density lipoprotein while raising levels of high-density lipoprotein.

On the other hand, exercise necessarily increases a person's rate of metabolism, as food is processed to provide energy expended through exercise. An inevitable side-effect of metabolism is the production of reactive oxygen species (ROS) and "free radicals" that can damage DNA and other cellular constituents. This cellular damage can lead to either cancer or accelerated aging due to cell senescence and cell death.

The paradox, then, is that the health benefits of exercise do not seem to be canceled out by the side-effects of higher rates of metabolism. It's an important issue not just for humans who are trying to stay healthy, but even more important in animals like birds that may need to expend energy continuously over significant periods of time.

So what's going on here? Perhaps this research has some of the answer:

The effect of marathon on mRNA expression of anti-apoptotic and pro-apoptotic proteins and sirtuins family in male recreational long-distance runners

Background

A large body of evidence shows that a single bout of strenuous exercise induces oxidative stress in circulating human lymphocytes leading to lipid peroxidation, DNA damage, mitochondrial perturbations, and protein oxidation.

In our research, we investigated the effect of physical load on the extent of apoptosis in primary cells derived from blood samples of sixteen healthy amateur runners after marathon (a.m.).

Results

Blood samples were collected from ten healthy amateur runners peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and bcl-2, bax, heat shock protein (HSP)70, Cu-Zn superoxide dismutase (SOD), Mn-SOD, inducible nitric oxide synthase (i-NOS), SIRT1, SIRT3 and SIRT4 (Sirtuins) RNA levels were determined by Northern Blot analysis. Strenuous physical load significantly increased HSP70, HSP32, Mn-SOD, Cu-Zn SOD, iNOS, GADD45, bcl-2, forkhead box O (FOXO3A) and SIRT1 expression after the marathon, while decreasing bax, SIRT3 and SIRT4 expression (P < 0.0001).

Conclusion

These data suggest that the physiological load imposed in amateur runners during marathon attenuates the extent of apoptosis and may interfere with sirtuin expression.

There are two main findings here, related to apoptosis and sirtuin expression. Let's take them in order.

Apoptosis is a form of programmed cell death that has several purposes. The invocation of a cell's apopotosis program isn't necessarily an indication that something is wrong. For example, it occurs normally during embryonic development. Early in the development process embryos of all tetrapods have tissues between what will become the fingers and toes of their hands and feet. But since animals that have left an aquatic environment are usually better off without this extra tissue, evolution has led to signals at a certain stage of embryonic development that cause apoptosis in the cells of the relevant tissue. This is an example of what's known as the "extrinsic" apoptotic pathway.

But for our present purposes there's a second pathway – the "intrinsic" pathway – which is used whenever a cell either detects internal damage (usually to its DNA) or some stressful condition, such as an excessive level of reactive oxygen species. A ROS is a chemically-reactive molecule containing oxygen, including what are sometimes called "free radicals".

This condition of excess ROS is called oxidative stress. It can occur for various reasons, including exposure to high levels of heat or ultraviolet radiation – or abnormally rapid cell metabolism due to vigorous exercise. Cells recognize the condition of oxidative stress indirectly though signaling involving various other molecules that are produced in response to particular ROS molecules. Among such indicators are proteins called heat shock proteins. Two members of this family that were measured in the research under discussion were HSP70 and HSP32.

Signals of oxidative stress trigger the second, "intrinsic" apoptotic pathway, which involves a cell's energy-producing organelles, the mitochondria. The main players in the intrinsic pathway are proteins called, generically, "caspases" – short for "cysteine-rich aspartate proteases". Caspases are enzymes that cleave proteins at aspartate units. (Cysteine and aspartate are two of the 21 amino acids that normally make up proteins.)

Caspases are fairly active enzymes, so they don't ordinarily occur at significant concentrations within cells. Instead, they are produced when needed from other protein enzymes called procaspases. One of these, procaspase-9 is found normally within mitochondria, along with another protein, cytochrome c. Most of the time these proteins are confined within the mitochondria. However, under certain conditions some channels in a mitochondrion's membrane can open and allow the release of procaspase-9 and cytochrome c. Once these proteins enter the cytosol (cell fluid) outside a mitochondrion, they can team up with another protein (Apaf-1: "apoptotic protease activating factor 1") to convert the procaspase-9 into the caspase known as caspase-9. The latter is an active enzyme that leads to the production of other caspases, with cell apoptosis as the eventual result.

Since a cell does not want to have apoptosis going on normally, the process must be tightly regulated. This is done (partly) by another pair of proteins, Bcl-2 and Bax. These two proteins have structural similarities and are considered to be in the same family, the Bcl-2 family. They are always present in the cytosol, and the relative concentration between Bcl-2 and Bax is what controls whether mitochondrial membrane channels will allow release of procaspase-9 and cytochrome c. If the ratio favors Bcl-2, the channels are essentially closed – the normal case – but if the ratio favors Bax, the channels open... and apoptosis may follow.

The present research measured the levels of certain proteins in 10 individuals before and after a marathon run. (The measurement was done indirectly by measuring levels of mRNA transcripts of the associated genes.) A key finding was that the ratio of Bcl-2 to Bax shifted in favor of Bcl-2 from the before to the after measurement. In other words, there was an anti-apoptotic effect, which countered the pro-apoptotic effects of ROS molecules produced by vigorous exercise. Although ROS levels were not measured (since there was no corresponding mRNA), levels of superoxide dismutase (SOD) antioxidants (Mn-SOD and Cu-Zn-SOD) increased after the marathons, reflecting ROS production.

Analysis of the results indicates that apoptosis actually was inhibited, though less in some experimental subjects than others. An increase in levels of procaspase-9 was not observed. Further, in 7 of the 10 experimental subjects, there was little evidence of DNA fragmentation (a consequence of apoptosis). In the other 3 subjects, there was some evidence of DNA fragmentation, but also smaller changes in the Bcl-2 to Bax ratios.

Most interestingly, there was a significant positive correlation in after marathon measurements between levels of Bcl-2 and both HSP70 and HSP32. This suggests that the expected increases of HSP70 and HSP32 may play some part in increased Bcl-2 levels. There was also a positive correlation post-marathon between HSP70 and Mn-SOD levels.

These findings, especially given the small sample size, certainly aren't conclusive. But, as the paper says, "Here, we have found a significant relationship between HSP70 and bcl-2 RNA ... following marathon, but the underlying cellular and molecular mechanisms involved in this [sic] exercise induced adaptations in apoptosis and HSP70 are unknown and require further investigation."

Expression of the sirtuins SIRT1, SIRT3, and SIRT4 pre- and post-marathon were also measured. (We've discussed the sirtuins on a number of occasions.) There's an extensive history of research on SIRT1, concerning its connections with such things as cellular metabolism, cell survival under stress, and antioxidant activity. Research on other sirtuins like SIRT3 and SIRT4 is less extensive. However, members of this family have various things in common. All are enzymes. SIRT1 and SIRT3 are histone deacetylases (HDACs), so have epigenetic roles in affecting gene expression. SIRT3 and SIRT4 occur in mitochondria.

Although it's possible to make various speculations about how sirtuins could be involved with apoptosis and metabolic consequences of exercise, not all that much is known about specific molecular mechanisms. Nevertheless, it's interesting that the present research does show an effect of strenuous exercise on SIRT1, SIRT3, and SIRT4 expression. The paper notes that "the RNA contents of SIRT1 increased substantially in the group after marathon.... On the other hand, the RNA contents of SIRT3 and SIRT4 decreased in the group after marathon."

Further research into these connections could be very interesting.

Marfe, G., Tafani, M., Pucci, B., Di Stefano, C., Indelicato, M., Andreoli, A., Russo, M., Sinibaldi-Salimei, P., & Manzi, V. (2010). The effect of marathon on mRNA expression of anti-apoptotic and pro-apoptotic proteins and sirtuins family in male recreational long-distance runners BMC Physiology, 10 (1) DOI: 10.1186/1472-6793-10-7

Further reading:

Running a marathon halts cellular suicide (5/11/10)


Articles related to sirtuins:

Sirtuin proteins (11/16/07)

The discovery of sirtuins, part 1 (11/17/07)

The discovery of sirtuins, part 2 (11/20/07)

Sirtuin news (1/21/08)

SIRT1 and cancer (10/26/08)

SIRT1 and cancer

In the past we've had some discussion of the histone deacetylase (HDAC) enzyme SIRT1 and other related sirtuin proteins, especially with respect to their possible relationship with longevity. (See here, for example.)

Much of the focus has been on the HDAC properties of SIRT1 that can switch off various genes. But there have also been findings of more direct relations between SIRT1 and cancer. Some indicate that sirtuins, including SIRT1, may help suppress cancer in certain circumstances, while others suggest it may actually help promote cancer. We'll have to save a general discussion of this relationship for later.

But now we have some research that shows how SIRT1 is directly involved, and has a beneficial effect, in an important pathway that's quite relevant to breast cancer.

The background is that the BRCA1 gene (short for breast-cancer-associated gene 1) is a tumor suppressor gene that, when mutated, may lose its ability to suppress tumors. Defective BRCA1 is sometimes inherited, which helps explain familial tendencies to breast cancer.

So what does BRCA1 normally do to suppress tumors? Well, apparently it maintains expression of SIRT1, which in turn inhibits the expression of another protein, called Survivin. The latter is an inhibitor of programmed cell death (apoptosis), and therefore, when it is active, helps protect cancer cells, which might otherwise be killed by the immune system, chemotherapy, or radiation.

In a nutshell: defective BRCA1 leads to insufficient SIRT1, which leads to an inadequate ability to kill cancer cells.

New Findings May Improve Treatment Of Inherited Breast Cancer (10/9//08)

About 8% of breast cancer cases are caused by mutations in tumor suppressor genes, such as breast cancer associated gene-1 (BRCA1). BRCA1 is the most frequently mutated tumor suppressor gene found in inherited breast cancers and BRCA1 mutation carriers have a 50-80% risk of developing breast cancer by age 70. "Although work with animal models of BRCA1 mutation has provided some insight into the many biological processes linked with BRCA1, very little is known about the downstream mediators of BRCA1 function in tumor suppression," says lead study author Dr. Chu-Xia Deng from the Genetics of Development and Diseases Branch at the National Institutes of Health.

Dr. Deng and colleagues were interested in investigating the relationship among BRCA1, SIRT1 and Survivin. SIRT1 is a protein and histone deacetylase involved in numerous critical cell processes including metabolism, DNA repair and programmed cell death, known as apoptosis. Although SIRT1 has been implicated in tumorigenesis, no concrete role in cancer initiation or progression has been identified. Survivin is an apoptosis inhibitor that is dramatically elevated in many types of tumors. Research has suggested that Survivin may serve to maintain the tumor and promote growth.

The researchers found that BRCA1 functioned as a tumor suppressor by maintaining SIRT1 expression, which in turn inhibited Survivin expression. When BRCA1 was not functioning properly, SIRT levels decreased and Survivin levels increased, allowing BRCA1-deficient cells to overcome apoptosis and undergo malignant transformation.

This leads one to ask whether there are other ways that SIRT1 activation could be maintained when BRCA1 is defective. Fans of resveratrol will observe that this is something that resveratrol can do. And so the researchers gave it a try:

They went on to show that the compound resveratrol strongly inhibited BRCA1-mutant tumor growth in cultured cells and animal models. ... In the current paper, resveratrol enhanced SIRT1 activity, this leading to reduced Survivin expression and subsequent apoptosis of BRCA1 deficient cancer cells.

Ironically, previous research had indicated circumstances in which SIRT1 might promote growth of other types of cancers. It might, for instance, inhibit expression of other tumor-suppressor genes.

Another news account goes into this a little more:

Gene thought to promote tumor growth has opposite role in a kind of breast cancer (10/9/08)

These results were surprising in light of previous reports showing that high levels of SIRT1 enhance growth of other types of tumors. It now appears that SIRT1 can enhance or inhibit tumor growth — it all depends on the context, says Deng. ...

The researchers also found that a red wine chemical called resveratrol, recently touted as a powerful antiaging compound, was effective in combating BRCA1-associated tumor formation specifically.

How resveratrol is able to do this is unclear. “The work in this case is that SIRT1 has an antitumor effect, and this paper provides mechanistic insights into that,” comments Pere Puigserver, a Harvard biologist who studies SIRT1. But the resveratrol data should be taken with caution, he notes. While this new research clearly shows the direct relationship between BRCA1 and SIRT1, the direct link between resveratrol and SIRT1 is more difficult to demonstrate.

Nonetheless, molecular details of BRCA1-related breast cancer are emerging, and this new data places SIRT1 squarely inside the complex web of molecules that impact tumor growth.

One of the main reasons that sirtuins are suspected of having cancer-promoting properties in some circumstances is that they may inhibit the highly important p53 tumor suppressor gene. (P53, when functioning properly, promotes cell apoptosis when DNA defects are detected during cell division.) In just one example of many, here's research from earlier this year that suggests a tumor-promoting property of sirtuins:

Switching on cancer killer gene (5/8/08)

Scottish scientists have discovered how to control a major anti-tumour gene that could lead to more effective chemotherapy. According to a report in the Cancer Cell Journal, research conducted by the Universities of St Andrews and Dundee may eventually lead to the development of new cancer drugs.

The gene, called p53 and known as "the guardian of the genome", is damaged or switched off in most cancers. But the resrchers found that they could reboot it using two new biological compounds called "tenovins".

In a laboratory study, the academics found that these compounds could kick-start p53 by turning off enzymes called sirtuins. Sirtuins act like genetic switches and keep p53 under control, ensuring that the cells stay alive.

Other news accounts of this research: here, here.

Tags: cancer, BRCA1, SIRT1, sirtuin, p53, resveratrol

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

More resveratrol hoopla

Resveratrol is in the news. Again.

My last major note about resveratrol is here, way back last September. How time flies. I also mentioned it more briefly here, in May, in connection with cancer. (Where its effect may involve facilitating apoptosis of tumor cells.)

But resveratrol's now back in the news again, so I guess it's time for an update.

As you recall, resveratrol seems to have a number of properties that confer health benefits. For example, it is thought to be an antioxidant, an anti-inflammatory, and perhaps to activate sirtuin enzymes, which may help produce an effect similar to calorie restriction.

The big question is whether you can get the benefits from the amount of the stuff you can get in a dose of reasonable size, for a reasonable price, and without having to drink gallons of wine per day (not a great idea).

Now we have recent reports of two more research results dealing with resveratrol. One suggests a benefit in countering obesity, and the other concerns anti-aging properties that mimic calorie restriction.

Here's the finding on obesity, the relation to which of resveratrol I cannot recall having heard bandied about before:

Red Wine's Resveratrol May Help Battle Obesity (6/16/08)

Resveratrol, a compound present in grapes and red wine, reduces the number of fat cells and may one day be used to treat or prevent obesity, according to a new study.

Past research found that resveratrol protected laboratory mice that were fed a high-calorie diet from the health problems of obesity, by mimicking the effects of calorie restriction. Researchers at the University of Ulm in Germany wanted to know if resveratrol could mimic the effects of calorie restriction in human fat cells by changing their size or function. The German team used a strain of human fat cell precursors, called preadipocytes. In the body, these cells develop into mature fat cells. ...

In the cell-based study, they found that resveratrol inhibited the pre-fat cells from increasing and prevented them from converting into mature fat cells. Also, resveratrol hindered fat storage.

One would certainly expect effects like that, if they can be reproduced in living humans, to be helpful in countering obesity. But there were two other beneficial effects as well:

[R]esveratrol reduced production of certain cytokines (interleukins 6 and 8), substances that may be linked to the development of obesity-related disorders, such as diabetes and clogged coronary arteries. Also, resveratrol stimulated formation of a protein known to decrease the risk of heart attack. Obesity decreases this substance, called adiponectin.

We've discussed both of these subjects before: IL-6 and inflammation were discussed here, while adiponectin was discussed here and here.

But the intriguing connections don't even stop there. Another report on the same research suggests that the effects related to fat cells may be mediated through sirtuin proteins:

Red wine component resveratrol might fight obesity, lab tests show (6/16/08)

Resveratrol’s mechanism of action is not entirely clear, but the compound seems to activate at least one member of a family of proteins called sirtuins. While also poorly understood, some sirtuins show up in fat cells.

Previous work showed that low levels of sirtuins allowed fat cells to add fats and to proliferate freely from nascent to mature stages, a recipe for weight gain. Conversely, that work also showed that an increase in sirtuins — in that case the compound Sirt2 — kept stem cells from maturing into full-fledged fat cells and inhibited mature fat cells from filling with fats.

In the new study, resveratrol’s good effects failed to emerge in either nascent or mature fat cells engineered to lack a sirtuin called Sirt1, Wabitsch said.

As potential therapeutics, “the sirtuins are a new class in the armamentarium of diabetes and pre-diabetes management,” says Henry Anhalt, a pediatric endocrinologist at Animas Corp. in West Chester, Pa., who wasn’t involved in this study. Sirtuins seem to curb the risk of obesity, cardiovascular disease and inflammation, all of which have been correlated with development of diabetes and its complications. The finding that resveratrol seems to work through a sirtuin (Sirt1) opens up new research opportunities, he says.

As previously noted, I've had a lot to say about sirtuins, which you can refer to here.

The second recent study, which appeared about two weeks before the one just discussed, involved experiments with mice that explicitly compared the effects of resveratrol and calorie restriction:

Substance In Red Wine, Resveratrol, Found To Keep Hearts Young (6/4/08)

[T]he researchers report that low doses of resveratrol in the diet of middle-aged mice has a widespread influence on the genetic levers of aging and may confer special protection on the heart.

Specifically, the researchers found that low doses of resveratrol mimic the effects of what is known as caloric restriction - diets with 20-30 percent fewer calories than a typical diet - that in numerous studies has been shown to extend lifespan and blunt the effects of aging.

This research sharpens results that have previously been found, and also shows that the required dose of resveratrol may not be unreasonable:

Previous research has shown that resveratrol in high doses extends lifespan in invertebrates and prevents early mortality in mice given a high-fat diet. The new study, conducted by researchers from academia and industry, extends those findings, showing that resveratrol in low doses and beginning in middle age can elicit many of the same benefits as a reduced-calorie diet.

"Resveratrol is active in much lower doses than previously thought and mimics a significant fraction of the profile of caloric restriction at the gene expression level," says Tomas Prolla, a UW-Madison professor of genetics and a senior author of the new report.

Another way this research differs from earlier work is that it looks specifically at the expression of genes known to be affected by aging in several important tissue types:

The group explored the influence of the agent on heart, muscle and brain by looking for changes in gene expression in those tissues. As animals age, gene expression in the different tissues of the body changes as genes are switched on and off.

In the new study - which compared the genetic crosstalk of animals on a restricted diet with those fed small doses of resveratrol - the similarities were remarkable, explains lead author Jamie Barger of Madison-based LifeGen Technologies. In the heart, for example, there are at least 1,029 genes whose functions change with age, and the organ's function is known to diminish with age. In animals on a restricted diet, 90 percent of those heart genes experienced altered gene expression profiles, while low doses of resveratrol thwarted age-related change in 92 percent. The new findings, say the study's authors, were associated with prevention of the decline in heart function associated with aging.

Another report stresses the overlap between the effects of calorie restriction and of resveratrol:

Red wine compound seen protecting heart from aging (6/4/08)

Using a method that permits simultaneous analysis of thousands of genes at the same time, the researchers found a huge overlap in the genes whose activity were changed by resveratrol and caloric restriction.

They looked at the heart, brain and muscles, and said that the effect of resveratrol was strongest in the heart but did prevent some aging-related changes in the other tissues.

A similar news release on this research mentions an upcoming Phase I human clinical trial that will study the effects of resveratrol on older humans:

Substance in red wine found to keep hearts young (6/5/08)

Resveratrol is currently sold over-the-counter as a nutritional supplement with supposed anti-cancer, anti-viral, anti-inflammatory and anti-aging benefits, although few scientific studies have verified these claims in humans. That may soon change: Researchers at the University of Florida hope to explore the effects of resveratrol on older people in a phase 1 clinical trial, set to begin this summer.

The study will assess the supplement's effects on memory, physical performance, inflammation and oxidative damage.

It also calls attention to the possible longevity-promoting effects of resveratrol on the mitochondria of cells:

Mitochondria, the tiny power plants that keep a cell functioning, are especially vulnerable to the oxidative damage that accumulates during the aging process.

"In animal studies, (resveratrol) seems to promote mitochondrial health," said Todd Manini, also a principal investigator of the upcoming trial and an assistant professor of aging and geriatrics in the UF College of Medicine. "Mitochondria are everywhere: They're in the brain, in the muscle, the liver. So it could have kind of a global impact on many different organ systems."

New York Times science writer Nicholas Wade (who, in earlier articles, had questioned the necessary dosage of resveratrol, see here) has a cautionary article that puts this research into context of other work on resveratrol and sirtuins. Among other points, he notes that there is still plenty of room to question whether resveratrol, or something similar, will actually have health benefits in humans, for example:

New Hints Seen That Red Wine May Slow Aging (6/4/08)

Dr. Auwerx, who used doses almost 100 times greater in his treadmill experiments, expressed reservations about the new result. “I would be really cautious, as we never saw significant effects with such low amounts,” he said Tuesday in an e-mail message.

Another researcher in the sirtuin field, Dr. Matthew Kaeberlein of the University of Washington in Seattle, said, “There’s no way of knowing from this data, or from the prior work, if something similar would happen in humans at either low or high doses.”

More news reports about this:

• Red Wine Compound Helps Heart, May Slow Aging (6/5/08)
  
• Study boosts wine chemical hopes (6/4/08)
  

Update, 7/16/08: There's more recent news about resveratrol here.

Further reading:

A Low Dose of Dietary Resveratrol Partially Mimics Caloric Restriction and Retards Aging Parameters in Mice – abstract and complete technical article describing the mouse study

Low-dose resveratrol as a calorie restriction mimetic – 6/12/08 blog post with further comments on the mouse study and associated issues

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

Resveratrol and cancer

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

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

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

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

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

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

Laboratory experiments showed that resveratrol:

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

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

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


Tags: resveratrol, sirtuins, cancer

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