The discovery of sirtuins, part 2
Unless you're a biologist who's already familiar with the ins and outs of research into sirtuin proteins, you might want to have a look (if you haven't already) at the previous note in this series, where I describe a lot of important background and provide various other references.
But if you're ready to forge ahead, in this note I'm going to write about a gene found in the nematode Caenorhabditis elegans. The gene is called sir2-1, and it's a homologue of the yeast SIR2 gene. (I. e. the two genes have very similar DNA sequences.)
C. elegans and some of its genes (like sir2-1 and several others affected by it, as mentioned here and here) has been studied by many investigators, because it's an easily-grown model organism for many biological processes that occur in multicellular creatures. And that's in spite of its simplicity – adults have a grand total of only 959 somatic cells.
Sydney Brenner began research into the detailed biology of C. elegans in 1974. Brenner had already earned his scientific spurs for helping decipher the 3-letter DNA code in the 1960s. But the Nobel Prize he shared in 2002 was awarded for his work with worms – which is an impressive statement about the importance of that work. Other prominent names associated with research into C. elegans include Cynthia Kenyon and Gary Ruvkun.
However, for the initial study of SIR2-like genes in C. elegans, we can return to the laboratory of Leonard Guarente. Soon after he and others at the lab had begun to appreciate the importance of SIR2 for longevity in yeast, Guarente suggested to a postdoc in his lab, Heidi Tissenbaum, that it might be very rewarding to figure out whether there were similar genes in the nematode that played a role in longevity. Tissenbaum was a pretty natural choice for this project, since she'd just recently done her thesis work in Gary Ruvkun's lab.
Despite the simplicity of C. elegans, following up on this suggestion was more easily said than done. Some idea of the complexity involved can be gained from the fact that there are about 20,000 genes in the worm's genome, as had only very recently been realized, since C. elegans was the first animal to have its whole genome figured out. The worm has almost 90% as many different genes as a human.
Among these 20,000 or so genes were four that were like SIR2. Which, if any, of those might have longevity-prolonging effects? Guarente and Tissenbaum set about trying to answer the question. So as not to miss any genes that might affect longevity even though unlike SIR2, they did experiments that could turn up others among the 20,000. They did this by considering different strains of C. elegans, each of which had one random section of its DNA duplicated. Since a eukaryotic organism already has two copies of each gene, this meant each strain would have 50% more copies (3 instead of 2) of the genes on the duplicated segment.
They found only one out of the 40 strains they tested that had a significantly longer life span. And the duplicated section of DNA contained only one of the 4 known SIR2-like genes – sir2-1, which was also the one closest in sequence to SIR2. Talk about "things that make you go hmmmmmm..."
To further strengthen the evidence that sir2-1 was somehow responsible for the increased life span, Tissenbaum produced a strain of C. elegans whose only extra gene was one or more extra copies of sir2-1. Lo and behold, these worms indeed lived much longer.
That's all well and good, of course. But how does sir2-1 bring about this increased life span? It certainly couldn't be much like the way SIR2 works in yeast to raise longevity. As you recall, longevity in a yeast cell is measured by how many times it is capable of budding off daughter cells. Normally, this is about 20 times. But this number can be substantially increased in a yeast strain with extra copies of SIR2.
However, the biology of C. elegans is quite different. The life span of these worms is manifested in a very different way than by how often cells are capable of dividing. In fact, the cells of an adult nematode do not divide at all – they have reached a state known as "senescence", all 959 of the somatic cells. All the difference in life span of a nematode occurs after its cells become senescent.
Initially, life span of the worms was measured simply by how long it took before the creature stopped wriggling, about 20 days. Later, more careful observation showed that aging could actually be noticed visibly (under a microscope). Old worms looked wrinkled and exhibited other visible signs of decrepitude. This is of importance, because an alternative hypothesis about how sir2-1 promoted longevity was that it somehow blocked a disease state that could kill the worm. But in fact, it was found that extra sir2-1 genes indeed slowed the rate of visible aging.
So there still remained to find an account of how sir2-1 extended life span. There were several other worm genes that were already known to affect longevity. I noted two of these (daf-2 and daf-16) here. Some of this information was already known to Guarente and Tissenbaum. In fact, the latter herself had participated in some of the relevant research while working in Ruvkun's lab. This 1997 press release describes some of that research:
Inactivation Of Key Gene Allows Worms To Develop Without Insulin (10/29/97)
(If you're confused by the capitalization of daf-16 and DAF-16, note that the former refers to the gene, and the latter to the corresponding protein. But you're not alone, since the opposite convention is sometimes used.)
I suppose that, at this point, the suspense is killing you, or at least delivering a credible threat to curtail your life span, so I'll just summarize what has been learned over the years about daf-2, daf-16, related genes, and how sir2-1 fits into the picture.
The hormone insulin plays an important role. In mammals insulin has a signaling function that stimulates cells to take up glucose and metabolize it. However, its role in C. elegans is somewhat simpler. There it doesn't directly affect glucose metabolism, but it still acts as a signal, as a trigger of the so-called "insulin-signaling pathway". This pathway keeps the daf-16 gene turned off as long as a cell-surface receptor detects insulin.
The protein coded for by daf-16, namely DAF-16 (duh), is a transcription factor, which means it enables the expression of other genes. When this happens in an immature worm, the result is an alternative developmental path, in which the worm enters a larval state, called a "dauer" (German for "enduring"). (The name "daf" is short for "dauer formation".) A dauer will eventually, after some delay, develop into a normal adult anyhow. But evolution has provided this dauer stage in case times are lean, and a delay will allow the organism to survive a little longer, on the hope that better times will come soon.
Under normal conditions, when sufficient nutrients are available, insulin is produced. A cell surface receptor (DAF-2, coded for by daf-2) detects the insulin and initiates a signaling cascade within the cell, and this in turn keeps daf-16 inactive. This does no harm to the organism, and in fact worms get along just fine even without a daf-16 gene, assuming adequate nutrition.
However, assuming insufficient nutrients, insulin levels drop. If that happens early enough in the nematode's life, daf-16 becomes active and triggers the dauer state. But what occurs after the nematode reaches adulthood and daf-16 becomes active (due to low insulin level) is perhaps even more interesting: the worm's aging slows down, and total life span increases. So this is a second way that the worm, even after it reaches adulthood, may be able to survive when food runs low, in the hope for better times.
Why isn't this second scenario simply the normal one? Why bother with the dauer stage at all? The answer is probably that nature has found this "live fast and die young" strategy the most successful in the long run, just as with small rodents. After all, a C. elegans is pretty small – 959 cells and about 1 mm in length. It's easy prey to larger predators that can enjoy a nematode meal. On the other hand, in cases there's not enough food for the worm to "live fast", it's nice to have not one but two backup strategies.
So where does sir2-1 fit in to all of this? Well, just as with SIR2, the worm homologue produces a deacetylase enzyme that inhibits the production of other proteins. One or more of these proteins is a necessary part of the signaling cascade that insulin initiates to keep daf-16 inactive. So extra sir2-1 protein interferes with the insulin signaling and, in effect, activates daf-16, which slows down aging, and extends life span – even when adequate amounts of nutrients are available.
Pretty neat, eh? That's evolution for you – always coming up with the Rube Goldberg schemes.
OK, that's how sirtuins work in nematodes. What about mammals, like us? As you might suppose, since mammals have far more than 959 cells in their bodies, things are a lot more complicated. There are even (at least) seven different homologues of SIR2. But the fact that in worms sir2-1 messes with insulin signaling and metabolism is a clue. Those are pretty important processes in mammals too.
To be continued.
----------------------
Further reading:
daf-16: An HNF-3/forkhead Family Member That Can Function to Double the Life-Span of Caenorhabditis elegans (11/14/97)
Reproductive Signals Affect Lifespan In Roundworm C. Elegans, Offering Possible Insight Into Human Aging Process (5/27/99)
Smell, Taste May Influence Lifespan Of The Roundworm C. Elegans (12/17/99)
Long-Lived Worms (3/8/01)
University Of Colorado Researchers Identify Switch That Controls Aging In Worms (12/11/01)
Stem Cells For Eggs And Sperm Also Control Aging In Roundworm (1/18/02)
DAF-16 Target Genes That Control C. elegans Life-Span and Metabolism (4/25/03)
Scientists Find What Type Of Genes Affect Longevity (7/1/03)
Old Worms, New Aging Genes (8/2/03)
Methuselah Worm Remains Energetic for Life (10/27/03)
Signs Of Aging: Scientists Evaluate Genes Associated With Longevity (4/18/05)
For The First Time: Longevity Modulated Without Disrupting Life-sustaining Function (3/11/06)
Eat Less, Live Longer? Gene Links Calorie Restriction To Longevity (5/2/07)
Genes That Both Extend Life And Protect Against Cancer Identified (10/15/07)
----------------------
Tags: sirtuins, aging, longevity, calorie restriction, caloric restriction
But if you're ready to forge ahead, in this note I'm going to write about a gene found in the nematode Caenorhabditis elegans. The gene is called sir2-1, and it's a homologue of the yeast SIR2 gene. (I. e. the two genes have very similar DNA sequences.)
C. elegans and some of its genes (like sir2-1 and several others affected by it, as mentioned here and here) has been studied by many investigators, because it's an easily-grown model organism for many biological processes that occur in multicellular creatures. And that's in spite of its simplicity – adults have a grand total of only 959 somatic cells.
Sydney Brenner began research into the detailed biology of C. elegans in 1974. Brenner had already earned his scientific spurs for helping decipher the 3-letter DNA code in the 1960s. But the Nobel Prize he shared in 2002 was awarded for his work with worms – which is an impressive statement about the importance of that work. Other prominent names associated with research into C. elegans include Cynthia Kenyon and Gary Ruvkun.
However, for the initial study of SIR2-like genes in C. elegans, we can return to the laboratory of Leonard Guarente. Soon after he and others at the lab had begun to appreciate the importance of SIR2 for longevity in yeast, Guarente suggested to a postdoc in his lab, Heidi Tissenbaum, that it might be very rewarding to figure out whether there were similar genes in the nematode that played a role in longevity. Tissenbaum was a pretty natural choice for this project, since she'd just recently done her thesis work in Gary Ruvkun's lab.
Despite the simplicity of C. elegans, following up on this suggestion was more easily said than done. Some idea of the complexity involved can be gained from the fact that there are about 20,000 genes in the worm's genome, as had only very recently been realized, since C. elegans was the first animal to have its whole genome figured out. The worm has almost 90% as many different genes as a human.
Among these 20,000 or so genes were four that were like SIR2. Which, if any, of those might have longevity-prolonging effects? Guarente and Tissenbaum set about trying to answer the question. So as not to miss any genes that might affect longevity even though unlike SIR2, they did experiments that could turn up others among the 20,000. They did this by considering different strains of C. elegans, each of which had one random section of its DNA duplicated. Since a eukaryotic organism already has two copies of each gene, this meant each strain would have 50% more copies (3 instead of 2) of the genes on the duplicated segment.
They found only one out of the 40 strains they tested that had a significantly longer life span. And the duplicated section of DNA contained only one of the 4 known SIR2-like genes – sir2-1, which was also the one closest in sequence to SIR2. Talk about "things that make you go hmmmmmm..."
To further strengthen the evidence that sir2-1 was somehow responsible for the increased life span, Tissenbaum produced a strain of C. elegans whose only extra gene was one or more extra copies of sir2-1. Lo and behold, these worms indeed lived much longer.
That's all well and good, of course. But how does sir2-1 bring about this increased life span? It certainly couldn't be much like the way SIR2 works in yeast to raise longevity. As you recall, longevity in a yeast cell is measured by how many times it is capable of budding off daughter cells. Normally, this is about 20 times. But this number can be substantially increased in a yeast strain with extra copies of SIR2.
However, the biology of C. elegans is quite different. The life span of these worms is manifested in a very different way than by how often cells are capable of dividing. In fact, the cells of an adult nematode do not divide at all – they have reached a state known as "senescence", all 959 of the somatic cells. All the difference in life span of a nematode occurs after its cells become senescent.
Initially, life span of the worms was measured simply by how long it took before the creature stopped wriggling, about 20 days. Later, more careful observation showed that aging could actually be noticed visibly (under a microscope). Old worms looked wrinkled and exhibited other visible signs of decrepitude. This is of importance, because an alternative hypothesis about how sir2-1 promoted longevity was that it somehow blocked a disease state that could kill the worm. But in fact, it was found that extra sir2-1 genes indeed slowed the rate of visible aging.
So there still remained to find an account of how sir2-1 extended life span. There were several other worm genes that were already known to affect longevity. I noted two of these (daf-2 and daf-16) here. Some of this information was already known to Guarente and Tissenbaum. In fact, the latter herself had participated in some of the relevant research while working in Ruvkun's lab. This 1997 press release describes some of that research:
Inactivation Of Key Gene Allows Worms To Develop Without Insulin (10/29/97)
The team — which also includes first author Scott Ogg, PhD, Suzanne Paradis, Shoshanna Gottlieb, PhD, Garth Patterson, PhD, Linda Lee, and Heidi Tissen baum, PhD — discovered that insulin may control metabolism via inactivation of a second gene, daf-16. The researchers found that, although insulin normally is required to regulate metabolism in the worm C. elegans, as in humans, the animal no longer needs insulin if it also carries a mutation in daf-16. This gene encodes a DNA-binding protein that passes along insulin signals within the cell to control the production of enzymes that metabolize sugars and fats. The team proposes that in the absence of insulin, the DAF-16 protein becomes unregulated, and that its runaway activity may be the key cause of metabolic disease in diabetes. In support of this model, the research team shows that metabolic defects in worms with defective insulin signaling are "cured" by the inactivation of the daf-16 gene.
(If you're confused by the capitalization of daf-16 and DAF-16, note that the former refers to the gene, and the latter to the corresponding protein. But you're not alone, since the opposite convention is sometimes used.)
I suppose that, at this point, the suspense is killing you, or at least delivering a credible threat to curtail your life span, so I'll just summarize what has been learned over the years about daf-2, daf-16, related genes, and how sir2-1 fits into the picture.
The hormone insulin plays an important role. In mammals insulin has a signaling function that stimulates cells to take up glucose and metabolize it. However, its role in C. elegans is somewhat simpler. There it doesn't directly affect glucose metabolism, but it still acts as a signal, as a trigger of the so-called "insulin-signaling pathway". This pathway keeps the daf-16 gene turned off as long as a cell-surface receptor detects insulin.
The protein coded for by daf-16, namely DAF-16 (duh), is a transcription factor, which means it enables the expression of other genes. When this happens in an immature worm, the result is an alternative developmental path, in which the worm enters a larval state, called a "dauer" (German for "enduring"). (The name "daf" is short for "dauer formation".) A dauer will eventually, after some delay, develop into a normal adult anyhow. But evolution has provided this dauer stage in case times are lean, and a delay will allow the organism to survive a little longer, on the hope that better times will come soon.
Under normal conditions, when sufficient nutrients are available, insulin is produced. A cell surface receptor (DAF-2, coded for by daf-2) detects the insulin and initiates a signaling cascade within the cell, and this in turn keeps daf-16 inactive. This does no harm to the organism, and in fact worms get along just fine even without a daf-16 gene, assuming adequate nutrition.
However, assuming insufficient nutrients, insulin levels drop. If that happens early enough in the nematode's life, daf-16 becomes active and triggers the dauer state. But what occurs after the nematode reaches adulthood and daf-16 becomes active (due to low insulin level) is perhaps even more interesting: the worm's aging slows down, and total life span increases. So this is a second way that the worm, even after it reaches adulthood, may be able to survive when food runs low, in the hope for better times.
Why isn't this second scenario simply the normal one? Why bother with the dauer stage at all? The answer is probably that nature has found this "live fast and die young" strategy the most successful in the long run, just as with small rodents. After all, a C. elegans is pretty small – 959 cells and about 1 mm in length. It's easy prey to larger predators that can enjoy a nematode meal. On the other hand, in cases there's not enough food for the worm to "live fast", it's nice to have not one but two backup strategies.
So where does sir2-1 fit in to all of this? Well, just as with SIR2, the worm homologue produces a deacetylase enzyme that inhibits the production of other proteins. One or more of these proteins is a necessary part of the signaling cascade that insulin initiates to keep daf-16 inactive. So extra sir2-1 protein interferes with the insulin signaling and, in effect, activates daf-16, which slows down aging, and extends life span – even when adequate amounts of nutrients are available.
Pretty neat, eh? That's evolution for you – always coming up with the Rube Goldberg schemes.
OK, that's how sirtuins work in nematodes. What about mammals, like us? As you might suppose, since mammals have far more than 959 cells in their bodies, things are a lot more complicated. There are even (at least) seven different homologues of SIR2. But the fact that in worms sir2-1 messes with insulin signaling and metabolism is a clue. Those are pretty important processes in mammals too.
To be continued.
----------------------
Further reading:
daf-16: An HNF-3/forkhead Family Member That Can Function to Double the Life-Span of Caenorhabditis elegans (11/14/97)
Reproductive Signals Affect Lifespan In Roundworm C. Elegans, Offering Possible Insight Into Human Aging Process (5/27/99)
Smell, Taste May Influence Lifespan Of The Roundworm C. Elegans (12/17/99)
Long-Lived Worms (3/8/01)
University Of Colorado Researchers Identify Switch That Controls Aging In Worms (12/11/01)
Stem Cells For Eggs And Sperm Also Control Aging In Roundworm (1/18/02)
DAF-16 Target Genes That Control C. elegans Life-Span and Metabolism (4/25/03)
Scientists Find What Type Of Genes Affect Longevity (7/1/03)
Old Worms, New Aging Genes (8/2/03)
Methuselah Worm Remains Energetic for Life (10/27/03)
Signs Of Aging: Scientists Evaluate Genes Associated With Longevity (4/18/05)
For The First Time: Longevity Modulated Without Disrupting Life-sustaining Function (3/11/06)
Eat Less, Live Longer? Gene Links Calorie Restriction To Longevity (5/2/07)
Genes That Both Extend Life And Protect Against Cancer Identified (10/15/07)
----------------------
Tags: sirtuins, aging, longevity, calorie restriction, caloric restriction
Labels: aging, Caenorhabditis elegans, calorie restriction, daf-16, longevity, sirtuin
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2 Comments:
Charles, since you posted this, I saw this article:
http://www.sciam.com/article.cfm?id=longevity-gene-caloric-restriction
It looks at Pha-4 protein which increased lifespan in worms, but MORE so if daf-16 production is blocked. I'm confused. The way you write makes the complex stuff understandable. Do you plan to work on a part III which might address this? Especially since there are human homologues to the Pha-4 encoding gene (foxa's). Thanks!
I'm planning to write more about longevity in C. elegans, but I don't know whether I can clarify what the article you mention says about daf-16. Need to look at what I can find in other references.
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