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)

Telomerase and Wnt signaling

Now that research into telomeres and telomerase has (finally) garnered a Nobel Prize, it's a good time to write about recent research on the subject.

Seminal work on telomeres by Elizabeth Blackburn, one of the Nobel winners, was published way back in 1978, and active studies have been going on ever since. So perhaps it's not surprising that the rate of new findings is not so rapid as occurs in newer areas – such as stem cells.

But fascinating new results on telomeres and telomerase do still appear, and one of them connects with more recent research areas – such as Wnt signaling and... stem cells.

Here's the press release:

Discovery pinpoints new connection between cancer cells, stem cells (7/1/09)

A molecule called telomerase, best known for enabling unlimited cell division of stem cells and cancer cells, has a surprising additional role in the expression of genes in an important stem cell regulatory pathway, say researchers at the Stanford University School of Medicine. The unexpected finding may lead to new anticancer therapies and a greater understanding of how adult and embryonic stem cells divide and specialize.

Don't bother getting excited about the "new anticancer therapies" bit. That's just boilerplate that about 77.3% of all press releases dealing with cell biology contain, presumably to impress the rubes. If you need something to get excited about, you might recall that telomerase is also being investigated intensively in connection with issues of aging and longevity, independently from cancer. However, while it's possible that something of medical significance may come from this research, that's probably way down the road.

Before discussing the new research, let's review some of the background on telomeres, telomerase, and Wnt signaling.

To begin with, a telomere is a series of short repeated segments of DNA found at the ends of chromosomes in all eukaryotic cells. In cells of vertebrate animals the repeated segment is TTAGGG (where the letters represent nucleobases: T=thymine, A=adenine, G=guanine).

The total number of nucleotides in these repeated segments varies a great deal from species to species, but in humans it is (initially) about 10,000 nucleotides (or ~1700 complete segments). I say "initially", because part of the telomere is lost every time a cell divides – perhaps 50 to 200 nucleotides per division. Obviously, this means that an adult cell newly derived from a stem cell can divide at most 50-200 times before the telomere is all gone. In practice, the number is a lot less than the maximum, perhaps 40 to 60 times, which is called the Hayflick limit. Cells are programmed to stop dividing on reaching this limit, since otherwise useful DNA would be lost or damaged upon further division.

Why does this loss occur? It seems to be somewhat of an accident of the nitty-gritty details of how DNA replication occurs during cell division. I won't go into that, since it's best explained with some diagrams; you can read about it at Wikipedia. In fact, in the early days of molecular biology (around 1972), what happens at the end of chromosomes during DNA replication was rather puzzling, and the puzzle was called the "end replication problem". Now it's pretty well understood, though somewhat messy.

In any case, the loss of DNA from the telomeres during cell division is a fact, and it conveniently explains the existence of the Hayflick limit, which had been recognized since 1965. What happens when the teleomere is all used up and the limit is reached? Cells that have reached the limit don't necessarily die (though they might), but they do stop dividing, and they enter a static phase of cell life known as senescence.

If you think about it, senescence can be a problem, especially in certain tissues that need to continually replenish their cells, such as skin and the lining of the intestines, as well as hair, fingernails, etc. How is senescence circumvented in such tissues? The answer is (adult) stem cells. It turns out that stem cells are not subject to the Hayflick limit. It's not clear whether they are subject to limits at all.

Like any other cell type, stem cells also divide (by the process technically known as mitosis). But there is one difference from the way "ordinary" cells divide. Stem cells can divide asymmetrically, where one daughter cell is another stem cell, but the other daughter is a "progenitor cell", which is close to a normal, fully-differentiated adult cell of a fixed tissue type. Progenitor cells differentiate further into the final form when they divide, and they are able to divide only a limited number of times.

So how is it that stem cells are able to escape the Hayflick limit, dividing an indefinite number of times, even though they are also subject to the same loss of telomere nucleotides with each division? The answer is the enzyme telomerase, which is able to rebuild shortened teleomeres. It's fortunate for the longevity of complex organisms that telomerase exists, otherwise stem cells would not be able to divide often enough to allow tissues exposed to harsh conditions (such as skin and intestinal lining) to be replenished.

In order to explain the results of the research to be described here we need to say more about telomerase, but first let's summarize the function of telomeres. At first it may seem that all they do is compensate for the sloppy way that DNA replication works, by providing long stretches of expendable DNA at the ends of chromosomes. But there is one other notable function: teleomeres are a useful "cap" at the end of a chromosome that is recognizable to cellular mechanisms responsible for detecting DNA damage. If it weren't for the telomeres, the ends of chromosomes would be indistinguishable from DNA damage, which can result from errors in the replication process, as well as damage due to external agents such as ionizing radiation or harmful chemicals. Cells have various mechanisms for repairing many kinds of damage, but even if repair isn't possible, other mechanisms exist that recognize the damage and prevent further cell division or cause programmed cell death (apoptosis).

Not all DNA damage can be repaired or compensated for by apoptosis or cessation of cell division. Unrepaired DNA damage (however it occurs) is the main cause of cancer (though not the only one). So the existence of the Hayflick limit as a result of teleomere shortening acts as one defense against cancer. Cancer can be defined as the uncontrolled proliferation of cells as a result of DNA damage (affecting existing mechanisms that normally control proliferation) or other causes. So fixed limits on the number of times a cell can divide is one of a number of mechanisms organisms have to guard against cancer.

Before moving on, here's a quick summary of the functions served by telomeres: (1) compensate for the chromosome "end replication problem"; (2) make it possible for cells to distinguish chromosome ends from damaged chromosomes; (3) provide natural limits to the number of times ordinary cells can divide, as protection against cancer.

As noted above, the third of these functions is a problem for stem cells that do need the ability to divide an indefinite number of times. Repair of tissues exposed to harsh conditions is not the only circumstance this ability is needed. Another very important case is that of embryonic development. Multicellular, sexually-reproducing organisms start from a single cell (zygote). Yet there are close to 10¹⁴ cells in an adult human.

It is true that a single cell that underwent 47 cycles of cell division could theoretically produce that many cells. But that's really pushing the limits, since some cell types are needed in much larger numbers than others. The bottom line is that embryonic development is the other main circumstance when limits on cell division need to be overcome.

Telomerase is what makes this overcoming of telomere limits possible. And it does it in a pretty straightforward way. Telomerase is a complex molecule with three distinct parts. Two of these are proteins: Telomerase Reverse Transcriptase (TERT) and dyskernin, which are coded for by distinct genes. TERT does most of the work. The other part is a short piece of RNA, called the telomerase RNA component (TERC), which contains and is somewhat longer than the repeat unit (TTAGGG in vertebrates). Like any other reverse transcriptase enzyme (other examples of which occur in RNA viruses such as HIV), TERT simply translates a piece of RNA into DNA and inserts it into a chromosome. In the case of telomerase, the RNA is carried inside the enzyme complex itself. Telomerase does its job simply by replacing the telomere DNA that is lost from chromosomes during mitosis.

So telomerase performs the function of rebuilding telomeres, and this is essential in tissues where cells must proliferate rapidly, such as in embryonic development and tissue regeneration. But as we noted above, if the restriction on cell division is circumvented, the risk of cancer goes up. Although telomerase serves an essential purpose in specialized contexts, it also makes it possible for cells to become cancerous. Consequently, telomerase is not expressed in most adult body cells. However, telomerase is expressed in about 90% of tumor cells.

It might seem as though one approach to controlling or even destroying cancer could involve either inhibiting telomerase or developing a vaccine using telomerase to induce an immune response against telomerase-rich cancer cells. There are in fact various clinical trials exploring both techniques. But this is tricky and rather risky, because as we've observed, telomerase is needed in stem cells required for tissue regneration, at least once the cells have begun proliferating. Those cells need to be protected while they are simply doing their job – replenishing skin and intestinal linings, for example. We need to understand how such cells are controlled so that they work without leading to cancer.

Anyhow, TERT is a protein that is an essential component of telomerase, which plays an important role in cell proliferation. The new research we're finally almost ready to discuss shows that, surprisingly, TERT also plays a role in a completely different aspect of cell proliferation having nothing to do with telomeres. That's where Wnt signaling comes in.

Wnt signaling is a subject we've looked at several times before. Some of the relevant articles are here, here, here, and here. Wnt was first noticed in connection with embryonic development and tissue regeneration. This article has many examples. The name Wnt alludes to a gene called "wingless", because the gene causes fruit flies to lack wings when the gene is mutated.

Recently Wnt's importance in stem cells has also received a lot of attention. Some of our discussion of that may be found here, here, here, and here. Wnt's relevance to cancer and cancer stem cells is often touched on in most of all the articles listed.

Basically, Wnt is the name of a family of proteins that play an important role in signals promoting cell proliferation, especially in the context of embryonic development and tissue renewal (skin, intestines, hair, and immune system cells). Since Wnt proteins promote proliferation, they also play a role in cancer.

The fact, then, that new research shows teleomerase can enhance Wnt signaling is signficant as a second, entirely separate route through which it plays a role in both normal stem cell function and cancer.

Here's the research abstract:

Telomerase modulates Wnt signalling by association with target gene chromatin (7/2/09)

Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/β-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/β-catenin signalling by serving as a cofactor in a β-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior–posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert^-/- mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/β-catenin signalling pathway.

That's a pretty good summary of the paper, but I imagine most people would like a bit more explanation of what's going on.

Previous research had disclosed some interesting "coincidences" involving stems cells and embryonic development, in which both telomerase and Wnt signaling seemed to have similar effects, even though no obvious connection was known. For example, epidermal (skin) stem cells spend most of their time in a quiescent (non-dividing) state. The main function of Wnt proteins is to carry signals between cells that inform target cells of a need to start dividing, for example during various stages of embryonic development or to heal wounds. The curious thing is that telomerase was known to have a similar effect as Wnt signaling on some stem cells. This coincidence is enough to motivate looking for a connection.

If you are really into this sort of thing, you might want to refer to this diagram of various cell signaling pathways, including that of Wnt. One of the things it illustrates is the position of β-catenin in the Wnt pathway. β-catenin operates at the end of the pathway, where it becomes a part of a protein complex that includes transcription factors (known as TCF/LEF), and the complex causes expression of a variety of Wnt-regulated genes, which go on to enable cell proliferation.

One of the main findings of the research is that TERT interacts with another protein called BRG1 (or SMARCA4), and together these become important parts ("cofactors") of the gene-regulating protein complex. It was also determined that TERT is the only component of telomerase that is involved with Wnt signaling.

Apparently TERT is more important for some instances of Wnt signaling than for others. For example, low levels of TERT in embryos of the frog Xenopus laevis resulted in very abnormal development of the frog embryos (in terms for the anterior-posterior axis of the body). But low levels of TERT at a later stage of development caused only somewhat more subtle defects in formation of ribs in the embryo. Similar somewhat minor effects also occured with low levels of TERT in mouse embryos, and these defects were much like the effects of low levels of β-catenin.

So what, then, is so interesting about this research? It's satisfying to know the reasons for what were previously just observed coincidences, such as the fact that telomerase is not only very important for the ability of stem cells to divide freely (during embryonic development and tissue regeneration) but that it also can help stimulate stem cell division.

But what makes this research especially significant is the importance of the biological processes in which both telomerase and Wnt signaling play major roles – namely embryonic development, tissue regeneration and repair, and cancer. The latter two processes are especially important for medical reasons, although we're still a long way from being able to use this knowledge about telomerase and Wnt signaling for therapeutic purposes.

As far as cancer is concerned, we need to understand much more about how telomerase and Wnt signaling work in various types of stem cells, given that stem cells may play a big role in some types of cancer (though not in others).

But tissue regeneration and repair are also of significant medical interest, since it is the eventual inability of various types of tissues to replenish themselves in old age that is responsible for the many debilities that appear in old age. Many people have speculated that telomerase could help alleviate this problem – provided it does not also lead to facilitating the development of cancer.

Finally, we are left with the puzzle of how it is that TERT happens to play important – yet quite distinct – roles in two very separate processes that are, neverthelesss, both important for cell proliferation. We don't know the answer to this, but we can speculate. Perhaps it's because TERT has to be around anyway for stem cells during embryogenesis and tissue regeneration. That being the case, perhaps at some point in evolution, TERT also happened to help boost Wnt signaling a little. The effect of amplifying Wnt signaling in the same contexts that telomerase was needed would be advantageous and worth conserving. This kind of double duty should lead to more efficient use of cell resources.

Park, J., Venteicher, A., Hong, J., Choi, J., Jun, S., Shkreli, M., Chang, W., Meng, Z., Cheung, P., Ji, H., McLaughlin, M., Veenstra, T., Nusse, R., McCrea, P., & Artandi, S. (2009). Telomerase modulates Wnt signalling by association with target gene chromatin Nature, 460 (7251), 66-72 DOI: 10.1038/nature08137

Further reading:

Cell biology: The not-so-odd couple (7/2/09) – expository article in Nature about the telomerase-Wnt research

Nobel in medicine honors discoveries of telomeres and telomerase (10/5/09) – news article in Science News

Nobel for insights into ageing and cancer (10/5/09) – news article in New Scientist

Chromosome protection scoops Nobel (10/5/09) – news article in Nature

Three Americans Win Physiology or Medicine Nobel (10/5/09) – news article at ScienceNOW.com

Work on Telomeres Wins Nobel Prize in Physiology or Medicine for 3 U.S. Genetic Researchers (10/5/09) – news article in Scientific American

Nobel Winners Isolate Protein Behind Immortality, Cancer (10/5/09) – news article at Wired.com

Tags:

telomeres, telomerase, Wnt signaling

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

Peroxisome proliferator-activated receptors and your ticker

Everyone knows by now that having diabetes raises the risk for heart disease in various forms. Wouldn't it be interesting to understand the biological reasons for this connection? Well, it turns out that various factors seem to be relevant, and one of them involves a variety of paths that all pass through the territory of a rather interesting protein called PPAR-γ.

Not only is PPAR-γ implicated in processes related to both diabetes and heart disease, but it turns out that some drugs used to control diabetes also affect the risk of heart disease – because of PPAR-γ.

We'll begin the discussion by looking at recent research on how PPAR-γ affects heart function.

First, of course, we should explain what a peroxisome proliferator-activated receptor is. The name is somewhat off-putting, and nowadays most people just write PPAR. It's also a bit of a historical artifact, in that this class of proteins was first investigated in connections with peroxisomes, which are cellular organelles that participate in the metabolism of fatty acids. That's an important clue right there, because if we are dealing with fat metabolism, there may well be connections with conditions such as obesity and cardiovascular disease.

It turns out that is only a part of what PPARs are connected with.

A PPAR is not a cell surface receptor. Instead it is a nuclear receptor, meaning that it's a protein found in the interior of cells that (like a surface receptor) is activated when it connects with hormones and similar molecules. Such molecules that bind to a receptor are called ligands.

Initially, the ligands in question were known to cause proliferation of peroxisomes, but now many other kinds of ligands that activate PPARs have been identified.

Once a PPAR has been activated it can affect the expression of many different genes, because it acts as a transcription factor.

Three important PPARs are known: PPAR-α, PPAR-β (also called PPAR-δ), and PPAR-γ. It's the last of these we'll be concerned with here. In fact, PPAR-γ seems to affect many cellular processes related to metabolism and other things. Recent research that we may discuss another time (see here) has shown that there are about 5300 sites in the DNA of a fat cell that PPAR-γ can bind to, and hence potentially affect the expression of nearby genes. So it's not surprising that PPAR-γ is involved in quite a lot of cellular business.

Incidentally, all three PPARs are produced from the same gene, with the variant forms being due to alternative splicing.

The research we want to highlight here deals with how PPAR-γ is linked with the daily rise and fall of heart rate and blood pressure. Such things that are part of the normal circadian rhythm, in animals, are usually regulated from the central nervous system. But that doesn't seem to be the only regulator:

What Makes The Heart 'Tick-tock' (12/2/08)

Researchers have new evidence to show that the heart beats to its own drummer, according to a report in the December issue of the journal Cell Metabolism. They've uncovered some of the molecular circuitry within the cardiovascular system itself that controls the daily rise and fall of blood pressure and heart rate. The findings might also explain why commonly used diabetes drugs come with cardiovascular benefits, according to the researchers.

"This is the first study to demonstrate that a peripheral clock plays a role in the circadian rhythm of blood pressure and heart rate," said Tianxin Yang of the University of Utah and Salt Lake Veterans Affairs Medical Center.

While much progress has been made over the years in understanding the body's master clock in the brain, the new study offers one of the first glimpses into the biological function of peripheral clocks in maintaining the circadian rhythms of tissues throughout the body, the researchers said.

There has already been reason to suspect that PPAR-γ is involved in this:

Earlier studies suggested a role for the nuclear receptor called peroxisome proliferator-activated receptor-γ (PPAR-γ) in clock function. PPAR-γ is perhaps best known as the molecular target for a class of widely prescribed and effective diabetes drugs called thiazolidinediones (TZDs), including rosiglitazone (trade name Avandia) and pioglitazone (trade name Actos). Those diabetes drugs are known to come with a side benefit: they have protective effects on the cardiovascular system.

The new research shows that the circadian variation in heart rate and blood pressure is disrupted simply by eliminating PPAR-γ from cardiovascular cells. The elimination was effected by working with two strains of mice in which suitable genes had been knocked out:

The researchers found that both knockout strains showed a significant reduction of circadian variations in blood pressure and heart rate. .... The mice also showed declines in variation of norepinephrine/epinephrine in their urine—a measure of activity of the sympathetic nervous system, which plays a key role in heart rate and blood pressure.

The animals had impairments in the rhythmicity of the major clock genes, including Bmal1, a transcription factor that controls the activity of other core clock components, they report. By treating the mice with the diabetes drug rosiglitazone, they were able to increase the activity of Bmal1 in the animals' aortas, the largest artery of the body that issues blood from the heart, and further study showed that the core clock gene is directly controlled by PPAR-γ.

What, more precisely, is the role of PPAR-γ in affecting rhythmicity? Apparently the effect is indirect, due to its abillity to activate Bmal1, which is known to be an important clock protein. This is indicated because rosiglitazone seems to be able to compensate for missing PPAR-γ.

Interestingly, other recent research has shown that the sirtuin protein SIRT1 also affects Bmal1. (See here.) this may be significant, since SIRT1 has gene-silencing effects that depend on nutritional factors.

What other processes is PPAR-γ involved with? Better-known than its effect on cardiovascular circadian rhythm is its role in fatty acid storage and glucose metabolism, and hence its connection with diabetes. But we'll have to look at that another time.

Further reading:

Protein Found to Set the Heart's Cadence (12/2/08) – Science News article

Tags: circadian rhythm