Hey everyone, welcome to the Peter Atiyah Drive. I'm your host, Peter Atiyah. The drive is a result of my hunger for optimizing performance, health, longevity, critical thinking, along with a few other obsessions along the way. I've spent the last several years working with some of the most successful top-performing individuals in the world, and this podcast is my attempt to synthesize what I've learned along the way to help you live a higher quality, more fulfilling life.
If you enjoy this podcast, you can find more information on today's episode and other topics at PeterAtiyahMD.com. In this podcast, I'll be speaking with my close friend and amazing scientist, David Sabatini. David's a professor of biology and a member of the Whitehead Institute at MIT. He's also an investigator at the Howard Hughes Medical Institute and a senior member of the Broad Institute, along with a bunch of other accolades that would take too long to get into here.
This podcast was actually recorded initially as part of an interview series I was doing for research around my book, and this was recorded in August of 2017. Maybe at some point we'll even just put the video up as this was actually done as a video interview with David, along with a number of his amazing postdocs, and certainly some of those will probably make their way into the podcast as well. Now, in this episode, we talk about his amazing journey in science and the work and stuff that he's done around mTOR and rapamycin, and if you've been following the blog and or paying attention to stuff that I'm interested in, you'll know that mTOR and rapamycin sit kind of at the heart of it. Now, about four years ago, David and I were having lunch one day, and it was kind of the first time that he ever really told me the full story of his work as a graduate student at Hopkins where he was part of the MD-PhD program, and I was just, you know, I remember sitting there taking notes on a napkin and thinking, God, this is such an incredible story of science, and I remember thinking, God, you know, one day we have to have this discussion again, but such that most people can actually hear it besides just me.
So part of what we discuss on the podcast is actually that journey and how as a young PhD, newbie, grad student, David methodically went after a problem that really wasn't even being particularly interesting at the time, which was to basically figure out how this thing called rapamycin actually worked, and of course, through the process ended up being the first person to identify this mechanistic target of rapamycin in mammalian cells. Now, the stuff that I find really interesting in this podcast is that David points out that he's, from an academic standpoint, kind of an unusual bird in that he's one of the few people who has carried his work from graduate school into his career, and that's actually pretty unusual. He's incredibly thoughtful, and some of you may have already heard a podcast that David, myself, and Nav Chandell, another good friend who will also be on the podcast, recorded back with Tim Ferriss on Easter Island back in the fall of 2016. We'll link to that here as well, and obviously, the reason we went to Easter Island was as sort of a pilgrimage based on the discovery of the bacteria that ultimately led to rapamycin, a bacteria by the name of Streptomyces hydrocofficus.
The interest I have in MTOR, of course, has to do with its central role in nutrient sensing, and of course, it's, I believe, and many believe, its central role in longevity. So, if you are interested in longevity, if you're interested in fasting, if you're interested in rapamycin, you're really going to want to listen to this podcast, because David is effectively MTOR man. I don't think there really is a person on the planet, and I'm saying that without trying to be hyperbolic, but I don't think there's anybody on the planet who knows more about rapamycin and MTOR than David Sabatini. And if you like this podcast, please make sure to check out the one that's going to be out soon with Matt Caberlin, which will take this discussion to another level as well, looking at Matt's work in dogs.
So, without further delay, here's my discussion and conversation with David Sabatini. Well, David, thank you so much for making time to sit down today and talk about what is potentially mutually our favorite topic of discussion. Before we jump into it, though, maybe for people who don't know you, can you tell us a little bit about how you got here and what did you do specifically? Sure, sure.
So, thank you, Peter, for coming and for visiting and both of you and for wanting to talk to me. So, I'm a biologist. I'm a professor of biology at MIT, and I'm also a member of the Whitehead Institute, which is where we are today. And I receive a lot of funding from the Howard Hughes Medical Institute, which is a key charity that works with biomedical researchers.
I have studied this protein that I'm glad you like a lot. It's my favorite protein called the MTOR protein, which is the protein through which this drug, rapamycin, which gets quite a bit of attention now, and I basically worked on that from the earliest point. We discovered that when I was a student. And so, my career as an MD-PhD, never really following the clinical track, though, and staying on the research side and finishing that and actually coming to the Whitehead in a program that is quite interesting and very unique at the time, which is that you can start your own lab after graduate school.
And so, I did that, and I eventually joined the faculty here, and now I've moved up the academic ranks. And to some extent, I'm a little bit strange from an academic point of view because I continue to work on, not exclusively, but to a large extent, what I started in my graduate school. So, most people, as you know, do something in graduate school, they do something in their post-doc, and then they sort of morph along the way. I kind of stuck with this mTOR protein, and in many ways, I was very lucky because we were there at the beginning, and it turned out to be such an exciting thing to work on.
So, let's go back to the beginning a little bit. You were an MD-PhD student at Hopkins, and after a couple of years of doing pre-clinical stuff, you pick a lab. Exactly, right. So, you do two years of medical school, and then you pick a lab.
And I was very fortunate to be taken by Saul and Snyder, who was, at the time, the head of neuroscience. He had a very big lab, lots of MD-PhDs. A lot of MD-PhDs wanted to go to his lab, and I was lucky that he let me go. And Saul is a really interesting man.
He still has a really prominent lab at Hopkins. In fact, the department is now named after him. And he was that person who had a lot of varied interests. So, he was a neuroscientist, but he was also a psychiatrist, and he was also a pharmacologist.
So, he really loved small molecules, and he loved particularly potent small molecules. That is, small molecules that act at low doses. How do we define a pharmacology as a small molecule? What's the kind of point?
You know, some people say 1,000 Daltons, which rapamycin is about that. The whole largest hand is sort of a non-peptide also, so it's not a piece of a protein. In many cases, it's not a natural molecule, although in our case, it's made by a micronutrient. It's not a natural to our body.
So, probably 1,000 Daltons. And so, he had this set of interests, and when I went to his lab, I was actually really interested in neuroscience. So, I had some classes in which I was sort of fascinated by some neuro questions. But when I got to his lab, I actually never did anything on neuroscience.
And I often told this story that the most influential scientific discussion I've ever had is when I went to talk to Saul. And basically, as a student, you need to pick a project. This is something that is quite challenging. I see my own students, they really get quite apprehensive about their projects.
So, I went to talk to Saul, and I went to his office. And I only met Saul, I don't know, maybe five or six times during my PhD. So, this was like a big deal. And so, I went to talk to him, and he basically said, David, we work on the brain.
And I thought that was great, because I wanted neuroscience. But then he didn't say anything else. So, that was it. And so, I remember leaving his office really anxious, because basically, I didn't have a project.
But I realized now, in retrospect, what he did is he actually was giving me complete freedom to do what I wanted to do. And that was, as I said, probably the most important thing I've ever done for me, because it really forced me to come up with my own project, and I think was a key sort of foundation in becoming the scientist I have. And it's something that I try to foster amongst my own people. So, anyways, I was in his lab, and I didn't have a project.
And at the time, they were actually working with this other drug called FK506, which is a... Simusipressin. It's used clinically still. Mechanism of action, although structurally, is very different than cyclosporine.
It actually mechanistically works on the same target, which is calcineurin. And at the time, this is before we had a lot of the tools we have now, like RNAi or CRISPR. And so, we needed controls. So, if you had a drug, what you tended to use was another drug that kind of looked like it, but didn't do the same thing.
And so, their control was ratomysin. And when I started reading about ratomysin... And this is what year? This would have been in probably late 91 to 92.
And it was clear to me that this molecule, in many ways, was much more interesting than FK506. And as you very well know, this had come from Wyatt Ayers, the pharmaceutical company, by Soren Seagal, who championed it. And there was a number of papers, which at the time were actually... A few papers were largely abstracts from meetings that show that it had antifungal effects, immunosuppressive effects, anti-cancer effects.
So, it seemed like an interesting molecule. I just come from medical school. We learned about immunosuppressants like cyclosporine, which at the time, you know, we're really just coming on, and we're really seen as miracles. But your lab's interest in FK506 was not its immunosuppressive properties, but its calcineurin inhibition.
Exactly. Because calcineurin, as the name implies, there's a lot in the brain. And so, in Saul's lab, they're basically studying the modulation of calcineurin in the brain using FK506 as a tool. And they were looking at cytotoxicity in the brain.
Things that, at the end, didn't lead, I think, in the directions they wanted to. But they were using it as a pharmacist, as a probe, basically. And so, I basically decided, why not try to work on rapamycin? And so, that's what I did.
And so, we... Which was just a control that nobody particularly cared for. Yeah, I think there were people in the world that were interested in rapamycin. But in your lab, yeah.
In your lab, yeah. People in the lab, no one was studying rapamycin. We had this great advantage, though, is that you couldn't buy rapamycin at the time. So, rapamycin was a compound that Wyeth was developing clinically.
It wasn't clinically available. You couldn't buy it. But Saul, being a very prominent scientist and having this interest in pharmacology, had actually written Seren Segal. And actually, he had sent us, probably without any of the legal needs that happens now.
Now, if you try to get a molecule out of a pharmaceutical company, the amount of paperwork and red tape is huge. But he had sent us a very significant amount of rapamycin, which I remember when it did start to be sold, which was equally expensive, I kind of back-calculated. It's a good value. Yeah, it was like millions of dollars.
Wow. Now, of course, it didn't really have that value, but theoretically, it was sort of millions of dollars, which, incidentally, that tube followed me all the way here. Did you still have the original tube? No, at some point, it was lost.
It disappeared at some point. But we actually had it, which was cool. So we could actually do experiments with it. And so I went on to try and try to work, and eventually, we purified this protein.
At the time, we actually called RAPT1, was the original name we gave it, and eventually, it was called RAPT1. And RAPT1 stood for? It was rapamycin and FKBTP target 1. And the reason that we, and also Stuart Shriver, who was at Harvard, and when he was working on this, he was also at Harvard, also identified mTOR, basically, at the same time.
He called it FRAP, which was FKBP, rapamycin-associated protein. And both of us were trying to accentuate the point that rapamycin acts with a co-receptor, this protein FKBP. From that point of view, it's a very unique kind of drug, where it doesn't directly bind to a protein target, but rather, it first binds to one target. And now, that drug receptor complex has a new surface on it, which now, in this case, interacts with mTOR.
And we were really trying to get that point across. Eventually, independently. Yeah, I had no idea. In fact, the only point where I found out they were working on it was once our paper had been accepted, I got missed with pretty lots of email.
We got a fax from a journalist saying he was writing an article on our paper and another paper from Stuart Shriver and actually sent us Stuart's paper, which we thought was really unethical at the time. And so we actually, at that point, contacted Stuart and said, hey, we got your paper. You should know we're working on this too, and here's our paper. So it was, yeah, I didn't know at all.
And in many ways, I was very naive, right? I was in this lab. Saul basically let us do whatever we wanted to. We had this drug.
Unbeknownst to Saul, I started working on this thing, right? And Stuart had a history of FKF-6, and that was a logical progression to what he was doing. Saul was not. It was funny.
He came from a world where people looked for the receptors for drugs. So if you look at his history, he'd really look for receptors for drugs, for small molecules, including endorphins, for example, that I worked with. But he wasn't big on cloning, what we call cloning a gene, which is where you have that get-the-DNA sequence. He almost thought you didn't need to do that.
Once you purified it, you could study the protein. So I was one of the first people there to actually clone a CDNA, as we call it, in his lab. So it was a fun time because it was clear that we got this protein. But you did this in a very short period of time because your paper, which was in Cell, was 1994.
1994, yeah. I worked like crazy. Really like crazy. And that lab in general worked like crazy.
It was very common to be there until 1 in the morning, and then I would usually show up at 7, 8 in the morning. We would sleep in the lab a lot. And once things started to go, so we were purifying. I purified it out of rat, out of rat brains.
And so we killed hundreds of rats to do this. My friends would help me kill them, take the brains out. There's a method in biology to visualize proteins called the silver stain, which is a very sensitive way of seeing a protein. And the first silver stain I did where I actually saw a glimpse of mTOR on this method.
I remember that really clearly because at that point, I knew I could do it. How did you know it was mTOR that you were looking at? Well, I mean, I had all the controls, and there was this band on what we call a gel that showed up just in the right place. And so I was like, okay, there is a protein here that has all the properties that I want.
And at that time, what properties did you know? You didn't know its size, did you? I didn't know its size. We know it bound to FKBP rapamycin.
But you didn't know that that exclusively bound to it, did you? We didn't. But we knew that it could be competed by FKF06 based on competition-type experiments, and so we had done that. So there was these features.
It was mostly the specificity that it required rapamycin to bind to FKBP. And that was crystal clear in the early experiments. When we had FKB by itself, there was no band on the gel. And when we added rapamycin, there clearly was.
And when we added FKF06, it clearly went away. And so we knew that that thing had all the right properties. But I remember very strongly feeling, OK, and at the time, now we have very, very sensitive methods to sequence proteins. Larger than mass per chromatin there, we didn't.
And so from what I saw in that gel to actually figure out what to sequence it was, I knew it was hard. But I knew it could happen. That was a very powerful feeling. It was the existing principle.
Exactly. So I knew the thing, like kind of the enemy existed, and I didn't get it. But then going from that initial glimpse on a gel to then having enough to actually sequence it, that's what took hundreds of rats to actually get to enough that I could purify it. And eventually we collaborated with this guy called Palm Tempts at Memorial Sloan Academy in New York, and he was able to sequence enough of the protein in the sense that I did a bunch of tricks and I got the whole thing at once, which also was kind of unheard of.
How did you do that? So back in the time, what people would do is they would get pieces and then they would sequence them and they would overlap and stitch them together. But what I did when I screened what we called libraries at the time for these pieces, I would get some pretty big pieces, but when I would sequence it, I knew that the front of the protein was missing, like I was missing, and I couldn't get it. I could never get beyond a certain point of the protein.
And so then what I did, which really turned out to be incredibly lucky, so what we would do is we would screen libraries of phages. And so this was basically people would take cDNA, complementary DNA, from rat brain, and they would clone it into these bacterial virus phages. And so now every little cDNA was in a virus, and you'd have hundreds of millions of this library. And you would plate it out on these plates, and a phage would make little plaques, and then you would screen those plaques.
So you'd have dozens of these plates, each with thousands and thousands of these little dots on them. And so what I decided to do is that I would screen this library with a piece that I knew was as far towards one end and as far towards the other end. So I screened it with both, and I looked for plaques that hybridized both. And in fact, when I first did it, it was so big that it was going to make the phage replicate slowly.
Because basically their genome was so much bigger now that to replicate, it would take longer. And on what order of times? It was probably two to three times more than it would take. So you could have been missing it.
I could have been missing it because the plaque that would have this would be incredibly small. And so what I did is I went back and redid it, and now I let the plaques grow longer. And I re-screened it, and in fact I got one plaque. It was a tiny, tiny little plaque that hybridized with both probes.
And when I looked at what was in there, it turned out to be the complete full-length cDNA, which was amazing because it was unheard of that these libraries would give you something like 9,000 base pairs. But it was. When I sequenced it, it was literally the intact thing from one end to the other. So I got very lucky because that would have been pretty hard to assemble at the time.
So you knew at the time, had Michael Hall's work in yeast been published yet? It had been published sometime during this period of time. But you didn't know anything. You didn't know even what the yeast form of this is.
No, when we started, when we first started getting sequenced, there was no sequence out there. And the yeast protein, really only the kinase domain is concerned. And so most of the peptide sequences that we had, that Paul Temps had sequenced for us, we didn't know at all where they were, right? And so these are kind of fun things that used to happen in the past.
You used to collaborate with a person who did protein sequencing and they would give you back a series of peptide sequences. But you didn't know what order they went. So let's say he gave you back, I think Paul gave me, Paul was amazing. He would give you, let's say, 15 peptide sequences.
He'd say, look, your protein, these 15 peptides exist in your protein. With or without overlapping those peptides? No, these are short. Mathematically, it's impossible to, by chance, figure out, like, you need more clues to figure out the order because it's combinatorially impossible.
Yeah, you have no idea Everyone that he said could be this or that, he was right, his prediction. So what you do is you have these peptide sequences. And what you could do now is design, we know the code, the amino acid code. So we can predict what the DNA sequence would encode.
But as you know, the DNA sequence is degenerate, right? So one peptide sequence can be encoded at the DNA level. You don't know what the assons and infarms look like. You don't know anything, right?
But each peptide could be encoded potentially by thousands of oligonucleotides. And you don't know the order of the peptides. What you would do is you would make a degenerate pool of oligonucleotides that have thousands of different ones. And you'd make them in both orientations.
And now you'd do PCR between them in all combinations. And you would find which was worked. And that would define the order of the peptides. And this is before you had real-time PCR.
Yeah, real-time PCR was used for quantitation. But we had PCR. And so we would take these oligolibraries and we'd mix and match them in all combinations in all orientations. And if you've got a band, it means that you've got it.
And then you could take those fragments and go into the libraries. And so it's funny because now, you know, with my students, when we discover a new protein, all you do is you look at the database because we have a whole genome sequence. I always tell my students that my paper, which was the discovery of M4, which at the time, to be fair, we did not realize how important M4 would be. My paper, basically, is like figure 1AB of their papers.
My whole paper is about the protein sequencing and all this kind of stuff. Was that paper effectively your PhD? That was my PhD. So you went back to finish a couple years in med school, obviously decided, I'm not going to do a residency, I'm going to become a full-time scientist.
And then you basically have been at MIT since or affiliated with MIT since. I was very independent. So people said, why don't you do one of these fellows positions where you can start your own lab? And at the time, there was only three.
There was the Whitehead one, there was one at Carnegie Institute, which is in Baltimore, and there was one at Cold Spring Harbor in New York. And I applied to all of them. And I got accepted pretty quickly, although after I graduated to Cold Spring Harbor and to Carnegie, that I didn't hear anything from the Whitehead. Like, nothing.
And only, like, basically, once I graduated, and I was kind of unemployed at that point, I was technically a post-doc in Sal's lab, but I hadn't taken, like, the boards, which Hopkins didn't make you take the boards, the medical boards to graduate, which was a nice thing. My mother was like, you're going to starve, you don't have a job, you can't do residency now because you didn't apply, you didn't take the boards. And then I got a call from Whitehead actually inviting me to interview, and I did. And then it took, again, a lot of time to, like, hear back.
And I remember they called me and said, look, we're going to offer you a position, but you need to understand you will never ever stay here as a faculty member, ever. I was like, okay. I realized I was applying for this Whitehead fellow position, not a faculty member. But then I came, and eventually I did stay.
And anyway, I look, actually, at history, they do keep about a third of the people who come through, but they give you this sort of speech that you will never ever stay. That's the expectation. And incidentally, many people named David have stayed. It's actually a good thing to be.
Actually, our current director was a Whitehead fellow. His name is David. One of the other faculty members named David. So I didn't know the time, but now I realize that David was a big advantage.
So how has your work evolved? I mean, you came here in the late 90s, right? In the late 90s, too. Rapamycin would go on to be approved by the FDA in 1999 as a frontline treatment as part of the double or triple cocktail for patients.
As rapimmune, right? Right, as rapimmune, along with often retinasomes, cyclosporine, or NMF. So now you're here. And I mean, we're going to get into much more detail, but effectively, you've never looked back.
You've never really left the space. I got here, and I was incredibly naive. I realized at this point how I thought, you know, I knew a lot. I thought I knew how to run a lab.
I had been very independent on my own. That doesn't mean that I was sort of independent from, like, running a lab. You know, behind the scenes in the small lab, I was doing the entire finances. I had written grants, the entire finances, organization.
There was a lot. Like, I could be independent, me, but in a lab is a different thing. So that was a hard transition to run, even though it was a small lab, to run a lab. And it was clear that at that time, I felt that this field had kind of plateaued.
There had been the discovery of mTOR, but we weren't getting very far. People were using rapamycin to look at lots of different things, and mTOR, by implication, through rapamycin was being connected to lots of different things. But one of the things that was obvious to me, and I think to others as well, was that mTOR had to act with partner proteins. And so we set about trying to identify what we now know are these mTOR-containing complexes, mTOR1, mTOR2, mTORcomplex1, and 2.
But again, it was really hard. We failed for years. It was, again, when this field has had a series of just, like, little things that until you figure them out, you make no progress. And so we would purify mTOR, and we'd look for other proteins.
We would continue to work upon them, so we just wouldn't find anything. To be clear, you knew that you had discovered the gene for TOR. Right. You suspected that this thing exists in different complexes.
And I already knew that there was other proteins, because when I was doing the mTOR original purification, the way that I was following mTOR was with a kind of a funky cross-linking assay, where I was cross-linking a radioactive FKBP to the putrid target, and there was always two bands on the gels. There was a protein mTOR, which I eventually purified, but there was a smaller one, which I could never get, either because it was just low abundance, I couldn't, I don't know what, but that little protein, which at the time I called RAF2, actually, basically remained unidentified. So I knew that there was something. So basically the first version of mTOR complex 1 was 4, and the version of mTOR complex 2 was RAF2.
No, no, no. That protein, actually, now that we found it, it turned out to be in both complexes. Oh, I see. But what I knew was that there was an associated protein with mTOR.
I knew from, I didn't know what its identity was, but it was very clear on all my experiments that there was a small, mTOR is very big, it's around 300 kW, which is a big protein. This was a little protein, it was around 30, so it was about 10 times smaller. So from a technical point of view, it's about 10 times harder to get, because there's about 10 times less peptides in that protein. So I failed to get it.
So when I got here to the Whitehead, I knew there was another protein to find, and we kept trying to go after this protein. And others, we knew it had to work, and it's a really big protein, big proteins work with friends. And it turned out, this is again, these little things, it turned out that the detergent, so when you work with mTOR cells, you have to lyse them, you have to break open the membranes, you typically use a detergent. It turned out the detergent we were using, which is by far the most common detergent that every lab in the world use, breaks apart these complexeses.
Just bad luck. And I had a postdoc, his name was Dostarvasov, who figured this out, and he found this other detergent called CHAPS. They kept them together. When you think back to your career, you're like, well, what are these key inflection points?
His discovery of that detergent was key, because once we did that, we purified all the interacting proteins, and that eventually led to mTORc1, mTORc2, eventually led to all the proteins associated with those. Basically, that was the key to all the biochemistry. There was several years of nothing, and he found that, and then everything has sort of, from that point on, we've sort of marched along in figuring out all the components of this pathway. We still don't know why things are sensitive to Triton.
We don't know why they're sensitive to Triton, but it's that kind of happenstance of science that, I guess, makes it interesting. So when, roughly by year, where are we when we have a, we meaning the world as a result of your discoveries in the lab, where are we when we sort of know that now we have mTORc1 around Raptor, mTORc2 around Rector? This is... It's around 2002, right?
So we were doing it around 2001, published around 2002. It's in that range. It's in the early 2000s. Although, as I said, we knew there was complexes even back in 94.
And at this point in time, your thought was, these two complexes control what, or sense what, or are important for what? Right. So it was very clear early on that mTORc1 was doing most of the things that we had connected before to mTORc. So we had rapamycin, and so rapamycin, in a way, had allowed us to know a lot about mTORc1, we now realize, than otherwise we would have known.
Because we didn't have really genetics, we didn't have easy ways of modulating mTORc, but we had rapamycin. And so there was a body of knowledge acquired by many different investigators about what was so-called downstream mTORc. What did mTORc do? We had some ideas.
It was a growth regulator, it regulates translation, it regulates autophagy, it regulates many, many metabolic pathways, it regulates cell size. We knew that largely through the use of rapamycin. And so now when we discovered mTORc1, which the first part we discovered was a protein called raptor, we now could go and say, well, does raptor matter for all those things? And it turned out it did.
So it was very clear that mTORc1 must be doing the things that we ascribe to rapamycin. mTORc2, therefore, remained very mysterious for a long period of time because it wasn't doing those other things. And only later did we find it was actually part of the PI3-kinase pathway in a regular AKT, and that clarified lots of things. And in many ways, mTORc2, you can actually even say, and we've written papers arguing this, it's almost like upstream of mTORc1 because the PI3-kinase pathway is one of the inputs into mTORc1.
In many ways, mTORc2 is less important than mTORc1. I mean, you can modulate it more and still survive more. So we really focused largely on mTORc1. And when I first got here, you sort of asked me, okay, what did you end up doing, right?
And I was pretty worked up when I got here and I had to realize I was sort of running a lab and unclear exactly what I was going to do. And I ended up working on mTORc1, or mTORc1, I should say, largely because I didn't know anything online. So I basically had to work on something. And I remember some people here were pretty critical of me working on rapamycin.
They were like, that's what I'm going to do. Even at the time, you didn't appreciate what you do now, which is that effectively, mTORc1 sits at the center of the universe for at least some of the things that we care a lot about, including potentially longevity. We did not. When did that become clear to you?
That became clear. We tried, when we started to understand the connection to nutrients and the fact that caloric restriction had been connected to longevity, we started thinking, okay, we actually tried to make experiments on worms at the time with rapamycin. Turns out rapamycin doesn't get into worms. But there was really some, there was an important paper in worms where there was a mutant in the C.
elegans version of mTORc1 that had longevity effects. I would say that was sort of the key paper. And this is unrelated to DAF2? Unrelated to DAF2.
Although, interestingly, in the screens that gave the DAF mutants, one of the DAF mutants, in retrospect, one of the ones that had never been identified what the gene was, was simply a mutant that had a mutation, turned out to be a raptor. I think it's DAF15. I don't quite remember. So there was...
It was 16, I'm sure. I don't remember. We'll look it up, yeah. But so it was interesting.
There was all these DAF mutants that had these interesting phenotypes. And once we found raptor, someone went back and found that one of the DAF mutants was actually a raptor. So that connected again to mTOR 1. Now, not only were the mutations in mTOR itself, in the C-Elegans mTOR, but also in the C-Elegans raptor that connected it to it.
We did not realize... Of course, our paper was published itself. Stuart Frye's paper was published in Nature. Emmer, Nature, Rode, and Use, and Views.
So people appreciated that defining mTOR mattered. But I think more from, okay, this is a new signaling pathway. This is a new component. I don't think we realized that it really...
We certainly didn't. At the center of so many important processes as we do now. People sometimes joke and say, well, mTOR does everything. So if something does everything, at some point, how interesting is it, right?
So it's a funny... Not a lot of people studying oxygen these days. Exactly. Or like from the ribosome.
We all appreciate the ribosome makes proteins and so it's important for everything. But you don't study it as a sort of something that's regularly... Although now we realize ribosome is very regular channel. Exactly.
So it starts to fall into that category. But luckily, we have enough of these regulatory systems that really shows us it's a very regular process. But today, mTOR and by extension, rapamycin and its analogs are really interesting, not just in your world, but in mine. So the plebes over here on the peanut gallery, this is super interesting, right?
This is potentially a molecule that could make people live longer, at least if what it does in yeast, flies, worms, and mammals is any indication. So why is it that rapamycin, or ask another way, why is it that the inhibition of mTOR, or specifically mTOR complex 1, as you'll probably elaborate on, can extend life? I find that a very interesting question and it's a question that I'm often asked and I think we say up front, we don't know the answer to that question. One way of addressing it is that you can eliminate many of the things that mTOR 1 does and then ask, well, now why inhibit mTOR 1?
Do I still get lifespan effects? If you do that and look at many different processes, probably you'd vote autophagy is the most important thing that it regulates, which as you know, autophagy is a self-eating process where the cell breaks down some of its own components and presumably has to remake them and so in a kind of naive way, you might imagine that what you're doing is throwing out the old and making new and again, naively, you might think, well, that's going to rejuvenate a cell, although none of that is of course proven. So that would be a simple answer, but it clearly is not the whole answer. So my answer to your question, why mTOR modulation has these longevity effects and yet many other pathways that in some ways are as complicated and as important for a variety of other things don't.
And this is the way I think about it. I think about it, I try to analogize it a little bit to a building. So if I want to take a building like this one and make it younger, rejuvenate it, I can't just get a plumber or an electrician or a painter or a carpenter because the building has many different features of which all of them have aged. What you really need is a general contractor who's going to then bring in all of those subcontractors and fix all the subsystems.
We look at an old building. An old building has lots of things that are messed up for it from the electrical systems to the windows, everything. And to some extent mTOR is like the general contractor for the cell. I don't know of any other pathway that does as many things.
mTOR basically has a finger in every major process in the cell. And so I think another way of thinking about your question is what's the simplest way to manipulate a cell so that lots of things are changed? And the answer to that is to modulate mTOR. Because all these other pathways will, you know, maybe some will regulate transcription, maybe some will do translation, some are going to change the shape of the cell.
But if you've got to do all those things, plus more, the only way of doing it with like a single hit is to go after mTOR. It is like the thing, it's like the brain of the cell which then has all these subroutines that do all these things. And so to me that's the simple answer is that to impact the state of a cell, to rejuvenate it, to slow the aging process, you can't do one thing, you can't do two things, you can't do three things, you can't do ten things, you can't do a hundred things. And the only way you can do all of those things with one button is to go after mTOR.
Now in biology, that tends to be a two-edged sword, right? Because presumably if you have one switch that controls so much, if you have the wrong general contractor or if the general contractor does the wrong thing, the effect is much more noticeable. So when did it become apparent to you or how is it apparent to you that this isn't just a linear relationship between signal and response? This is a very good point, right?
So you could say, well, it's a general contractor, there's a lot of things and so not only is anti-aging one of the things it does but how you sort of grapple sperm production which is a potential targeted heart function, right? All these things require it. So you might get the anti-aging effects but you're also going to get all the downsides. And I think that is certainly true and that's the major issue with targeting mTOR.
Because at the time you really kicked your efforts off here, people thought of rapamycin and mTOR as a one trick pony which was you give this drug every day, your immune system, specifically your cellular immune system doesn't work as well and at least for that subset of patients who had foreign organs in their body that's a reasonable thing to have. And incidentally, there is now, so if you find rapamycin started as an immunosuppressant, the interest in mTOR in the immune system pretty much was unexistent and now there's an entire field of so-called immunometabolism of which mTOR is probably 50% of the whole field and so it's mTOR 1, mTOR 2 in different immune cells, Tregs, right? T-halpers. How much of this came out of the Novartis work from three years ago?
Did this precede that? Well, it preceded that. I mean, the Novartis work was the first sort of work in humans, right? They clearly showed beneficial modulation in the immune system but in terms of studying which immune cells are most affected by rapamycin with the role of mTOR 1, that's come out of the academic role by a number of groups that were heavily enabled by the discovery of raptor and Richter because now you can genetically inhibit each of those and one of the things that my lab would really try to do is to put our mice out there and so people use, for example, our raptor mouse and flocks, so-called flocks raptor mouse a lot.
But this question of, in a way what you're saying is how much can we sort of tolerate mTOR modulation for beneficial effects versus non-beneficial ones? And again, and I don't think we have the answer to that. To some extent, rapamycin is not a complete mTOR 1 inhibition, we know that and complete mTOR 1 inhibition is probably not tolerated and so rapamycin might be as good as you can get and you get some modulation. I'll say a little bit more about that.
So you're saying if we could wave a magic wand, Bobby very eloquently spoke about why inhibition of mTOR 1 leads to inhibition of mTOR 2 and what the temporal relationship of that might be but I don't think we got into this issue which is if I could wave a magic wand and completely inhibit mTOR complex 1 not lay a hand on complex 2 why wouldn't that be a good thing? Because mTOR 1 is probably required for the growth of any normal cell. So to make for a cell to basically make its organelles, to make its proteins, to divide mTOR 1 is probably an essential. So at that level it would start to mimic a crude chemotherapeutic agent that modulates it.
It becomes 5FU at a ridiculous dose or something that's going to basically slough off epithelial. out. Exactly. It falls basically atrophy of everything, anti-growth, and probably cell death.
And in fact, in many tissues, when you delete Raptor, it can be quite bad. That's the phenotype? Yeah. Again, epithelium in the gut, at least when we've looked, that's the thing.
I see. So I don't think there's two issues going on here. As Bobby surely told you, Rapamycin will also, with a longer time point, inhibit MTRK2, and that is potentially bad for glucosomeostasis. Yes.
The other issue is that Rapamycin doesn't fully inhibit MTRK1. So in an ideal world, you'd like to have, and what I mean by that is that MTRK1 probably has dozens of substrates, and Rapamycin only effectively inhibits some of them, not others. Including combo autophagy is relatively weakly modulated by Rapamycin. Why is that?
Because the substrates, what Rapamycin basically does is sort of occlude the substrate binding channel in MTRK1, and it's physically occluding and depending, probably, this is somewhat hand-waving, but there's some evidence to this. Probably the size of the substrate, if it's smaller, it might get easier, and it's not occluded. If it's bigger, it's going to get blocked. And so probably the key substrates in the topology pathway simply are not as effective because they get into the kinase domain of MTRK1 still.
By the way, is this issue different for any of the Rapalogs? No. They're all basically producing the same effect as Rapamycin. Some people might argue differently from that, but in my experience with them, they are basically like Rapamycin with maybe different PK, PD properties.
But from a mechanistic point of view, I wouldn't expect differences in this, and I haven't seen those differences. So in an ideal world, you might want a molecule that would inhibit all the substrates of MTRK1, not touch MTRK2. But not do it institutely. Not do it institutely, but also maybe not 200% inhibition.
So I'm not sure I would use that molecule to wipe out MTRK1. I would use it to bring down all the MTRK1 activity of all, to resource it to some extent, leaving MTRK2 intact. I think that's going to be very hard to do by targeting MTRK1 itself, because MTRK1 and MTRK2 share the same kinase domain, and so you can't go for the ATP binding site, which is most kinase inhibitors. MTRK is a kinase, protein kinase, like Kleebeck, for example.
They all go for the ATP binding site. So we're not going to do it for that. And so our view is that the way to accomplish that is not to go after MTRK1 itself, but to go after its upstream regulators. And the big benefit, in my view, of doing that is that you should be able to have something now that modulates all MTRK1 substrates, and you can also start to get tissue specificity, because these regulators vary in importance across tissues.
The aspect of this pathway that's kept our attention for two decades at this point is that MTRK1 is basically regulated by everything. Anything I do to the cell, whether I change nutrients, oxygens, pH, growth factors, osmotivitis... What's the direct effect of glucose and or insulin in MTRK1? It obviously plays an enormous role on complex too.
It seems to activate them, right? So through independent pathways. There seems to be a pathway through which insulin acts, and there seems to be a pathway through which glucose acts. And even the glucose pathway probably has several sub-branches to it.
I see. Which, again, teleologically makes sense, because if it's a nutrient sensor, it should be activated by nutrients, but it becomes very complicated now, because you have the same nutrient acting in completely different areas. And that's probably because you're looking at... In the cells that we use in culture, we can get both of these sensing systems where probably in vivo there's tissues that are going to care more about the insulin arm, there's tissue that care much more about the glucose arm, and there's something to care about both.
So if you think about being a peripheral tissue, let's say you're a cell somewhere in your leg, and you need to make a... A muscle cell. Let's say muscle cell. You need to decide whether you're in an anabolic state or a catabolic one.
So clearly there's things of use and all that. But let's say just in response to nutrition. You kind of want two pieces of information, right? One, you want to know that the organism that you live in as a whole is in a fed state.
You want to be a good member of the community. And that is reflected by things like insulin, which basically tells you the pancreas. It's a global metric. Pancreas, salt, glucose, we sent out insulin.
And the other one is you actually want to know that you have the nutrient that you need. You can have, like, central command telling you, hey, I got glucose, but if you don't have glucose, you can't do it. And so you really want, like, the central signal and you want the local signal. So I think one can interpret that the pathway senses both the nutrients...
So the amino acid can be a local... Right. The glucose molecule itself is local. For sure.
We know it is. Whereas the larger peptide can be sort of the central command. And now you can extrapolate that, too. There are many signals that are secreted in response to food, right?
Insulin just being one of them. And then there are many local nutrients. And now you can start to see the enormous complexity of the problem, right? And now you add a temporal component to it.
And now you actually add a concentration. And then you make things tissue-specific. So our view has been, if we can find the sensors of the nutrients, and that's what we focus on, so we focus In 2015, in the fall, you had these two papers that came out that looked at leucine, of course, huge interest, but also arginine. Leucine and arginine can get into itself very easily.
Do they passively diffuse in? There's transport. There's relatively straight forward. Okay.
In the cytosol, these amino acids bind to receptors that then downstream result in the activation of TOR, specifically in TOR complex 1. People have long talked about how branched-chain amino acids are important for building muscle. Specifically to be consumed in a workout was always sort of the rhetoric, presumably because that's a very catabolic time for muscle. It now seems that that makes sense, at least in the presence of what leucine's doing.
Do we think that the other two branched-chain amino acids are having any effect? At least when we look at the receptor we found for leucine, and then we look at the concentrations at which it might bind to other branched-chain amino acids, we don't think those affinities are relevant. Particularly valine is way too low. Isoleucine, maybe in some situations, could act with the receptor, but unlikely.
So in our hands, again, looking at a very molecular point of view, it really seems like leucine is the key one. And I don't think, you know, from talking to bodybuilders and looking at bodybuilding products out there, it does seem like leucine is the one that people focus on more than the usual ones. And tell me, the difference between leucine and arginine, and with respect to the signaling, is what? One way to sort of conceptualize Amtrak-1 is, it wants to drive an metabolism.
And what its goal is to detect when something's missing for that. So we tend to think of the pathway like when we turn it on, but probably its really key function is to turn off when something's missing. Let's say you're building a house, all of a sudden you run out of concrete, you want to turn off. All of a sudden you run out of wood, you want to turn off.
So the default is on? The default, when everything is there, is on. But it's built, it's organized in such a way that the removal of anything can turn off. It efficiently turns off.
Now, this is going to vary, obviously, between different tissues. And so the pathway evolved that it needs to detect leucine, and it needs to detect arginine, at least in most tissues. Now, why is that? They're both amino acids.
If you think about this during the course of evolution, you're an animal, they ate another animal, so you ate its muscle, you got protein. Why do you need to sense two different amino acids? And they're very structurally different, right? They're about as structurally different as you could get in terms of amino acids.
We don't have an answer to that. Why did evolution do that? Take these two amino acids. I mean, that's a phenomenal question.
I don't know enough about amino acids to know what the evolution of amino acids look like. I mean, a billion years ago, I assume we didn't have the same amino acids. No, we did. We did.
Most all forms of life have problems. So basically, from the beginning of when we had DNA to RNA to protein, we had the exact same amino acids. So that's even more of a mystery. Why in the heck did we...
Part of people in the lab that I'm sort of encouraging to look at other organisms, because the sensing part of the system is probably evolving quite quickly, because different organisms live in different environments. In fact, flies, we know already, don't care about arginine. They care about leucine, and it turns out they care about a whole bunch of other amino acids that we don't care about. What about yeast?
So yeast, in many ways, is the most mysterious, because yeast... So we don't know any sensors in yeast, and none of the sensors we have found are in yeast. And that's because yeast can make amino acids. So that's what I'm saying, yeast is very primitive.
You give it nitrogen, you give it carbon, it's going to make every amino acid. So things like leucine, which are essential to us, are not essential to yeast. They can make it. In what state do yeast cease to activate TOR, only in the absence of the essential elements?
So regulation of TOR is not as well-studied in yeast, because it's harder to detect the output. And so typically what people do is they change the nitrogen source, or they change the carbon source. And so my view is that yeast has to have a sensor of nitrogen, whatever that means, right? It's not so easy to understand what that means.
And a sensor of carbon, but not a sensor of individual amino acids. And as we find more sensors, so we now have... We've now connected the pathogenmethionine sensor, and we have a receptor for that. That yeast doesn't have that either.
And so I've actually also tried to encourage people to look for what might be a nitrogen sensor in yeast. For example, ammonia, which is a simple form of nitrogen. Maybe that's what's sensed. Maybe acetate is what's sensed for carbon.
But we don't know. So say more about methionine, because in the protein restriction literature, certainly one argument is that methionine restriction specifically could be beneficial if one believes that low IGF is beneficial. And we can talk about whether that's causally the case or not. Not even getting into the IGF binding proteins.
Where does methionine fit into TOR? Right. So methionine actually is a very interesting one. As you said, there's extensive literature on what's so-called methionine restriction having quite beneficial effects from glucose homestays.
It's actually quite very reasonable, as good as cloric restriction. And there are some papers in flies, genetic papers, that suggest that some of the methionine restriction effects go through the TOR pathway in flies. We got to this basically through a protein. We found a protein of unknown function.
And we probably forgot what it did. And it turned out to be a sensor of this metabolite called SAM, acidenosylmethionine, which is basically made by methionine. So it's actually quite interesting. It's a supplement with a variant of SAM-E.
Exactly, right. SAM actually has some pretty interesting clinical effects. I see some quite convincing data on antidepressive effects of SAM out there. So the sensor here is interesting because the other sensors we have directly bind leucine, directly bind arginine.
This one doesn't bind directly to methionine. It binds to a metabolite made by methionine, which is SAM. Which SAM, many things can feed into SAM, so it actually can integrate lots of signals. So this sensor basically behaves like the other ones.
As soon as methionine levels go down, SAM levels go down, the sensor therefore inhibits this pathway. So SAM would not be a longevity agent by the oversimplification that excess SAM would be akin to excess methionine, would be akin to failing to inhibit TOR. Exactly. So methionine restriction could be originally rescued by giving SAM.
And we actually know in the pathway that we built in cells that that's true. You can bypass methionine simply by giving SAM. So a molecule that could basically trick this sensor into thinking that SAM was not there would be a quite interesting one. I think methionine is probably the most interesting of these amino acids because if you fast an animal, methionine is the amino acid that drops the most.
And you look at all the amino acids in mice. So we should do some of this in humans. But it kind of makes sense. I can volunteer.
We could definitely profile. The reason probably is that arginine, you can make some, right? Your liver can make it. And then leucine is an amino acid that's an essential amino acid that to some extent is only used to make protein.
That's it. So when you fast, you start to break down your muscle and release leucine. Methionine is not only an essential amino acid that you use to make protein. And remember, the first amino acid of all proteins is methionine.
So by definition, every single protein has methionine. But it's also incredibly metabolically active through SAM and the so-called methionine cycle. So when you fast, you probably just can't generate enough methionine by breaking down your proteins to keep up with methionine demand. Well, you can for leucine.
So if you look at the blood of an animal, it's fast. Methionine is the number one drop to me to ask. Do we think that's true in autophagy in general? We mean autophagy.
If we put an animal into a state that induces autophagy, independent of color restriction, so for example, would we see the drop in methionine as a readout? You know, you might expect it to go up, actually, right? Because autophagy is going to break down protein. You might methionine.
Yeah, if you're not recycling. If you're not recycling. It depends. If you induce, in a state, for example, post-exercise.
I don't know what we know about the use of methionine in SAM, right? So SAM is used for methylation reactions, right? And there are hundreds of methylation reactions. SAM is the second most common cofactor in enzymes after ATP.
Everyone knows about ATP, and ATP is energy, and it's used in many, many, many reactions for phosphorylation, but SAM is the second most common one. So there are literally hundreds of proteins that use SAM. So maybe after exercise, a lot of SAM is used. I don't know.
It's an interesting question, right? But with fasting, methionine definitely plummets. SAM definitely plummets. And so we're now generating the right animal models to ask whether the sensor we have is involved in the effects of methionine restriction.
So we can basically knock it out and then do methionine restriction. And if the animal doesn't have the health benefits of methionine restriction, it means that this sensor, and by extension, M4-1, are the key mediators of methion restriction. So we'll see. So coming back to rapamycin specifically and all of its limitations, so we've established that you can't just take rapamycin all day every day because that experiment's been done.
That's the clinical utilization of it. Certainly the animal data have suggested and the human data have suggested that an intermittent dosing of rapamycin could produce a beneficial phenotype with respect to longevity specifically and also with respect to immune function. So if you had to guess based on triangulating these data, assuming no new drug came along that was going to selectively do some of the things that we discussed, how would one dose in an animal, or a human for that matter, rapamycin, to increase the odds in favor of longevity and against harmful side effects, which presumably the most obvious ones would be immune suppression and or glucose homeostasis disruption? Yeah, and also epithelial, particularly the GI epithelium.
So I think the intermittent approach is definitely the one that makes sense. Because if you buy the idea that you want to induce autophagy, which you know a lot of people, of course, like yourself, who study the effects of that, fasting also view that that's one of the goals of fasting is to induce autophagy. So if we basically want to chemically induce autophagy without fasting, I think the intermittent dose is what makes sense. You basically let, have an induction autophagy, a relatively weak one with rapamycin, but then let the system rebuild.
It's clear that both mTOR, you need just right amounts, right? You can't have too little, it's toxic. If you have too much, it's toxic. It's the same thing with autophagy.
If you remove autophagy, it's really toxic. So if you have too much autophagy, it's really toxic. Cycling, anabolism, catabolism might be the single most important thing to do. It might be, right?
And I think it's hard for us to know, but those intermittent dosing strategies, every other day feeding strategies, all point to that. And the genetics, where too much is bad and too little is bad, also point to that. So if you genetically inhibit this pathway by deleting rapture, if you're genetically activated by deleting these repressors called the tuberous rosa complex, both are bad. Both, in fact, in many tissues, like the muscle, give the same output.
There's an overlap in muscular dystrophy here, isn't there? So this may be a theoretical question, but when we think about the life-extending properties of rapamycin, do we believe that it is a result of delaying the clinical onset of disease? Let's use a disease where that tends to be more binary, like cancer. Obviously, cancer spends probably 70% to 80% of its time undetectable, but due to just the law of growth, it becomes detectable only at the end.
So do we think that, inasmuch as, say, taking these agents would allow you to live longer by not dying from cancer at the same period of time, does it delay the time it takes for cancer to become clinically detectable and or delay the demise of the animal once it has that cancer? I think specifically, in the case of cancer, rapamycin is, there's some situations where it has some decent activity, but in general, it's not a cytopoccalation. It's not going to kill a cancer. Once an organism has cancer, do you want to know if it's doing anything to prevent the development of cancer?
We don't know that well, and there actually has been some epidemiological data where people have compared cancer rates in transplant patients. Identical patients who are within that rap. FK506 versus rapamycin. And it's actually quite interesting, because, as you know, immunosuppression in general is associated with higher cancer rates, right?
The idea that you have less immune surveillance, that's not seen in rapamycin. So it is seen in FK506, it's not seen in rapamycin. And the argument has been that rapamycin itself has cancer cell autonomous effect. Independent of the immune modulation problem.
So you're presumably getting less immune surveillance because it's immunosuppressant, although, of course, that's not proven. But you're mitigating that by now directing the rapamycin. And they cancel each other out. And you know the size of the effect from the FK506 cohort.
Exactly. And other immunosuppressant things like the sporens have also been looked at that. So my bet would be that in the case of cancer, you're not going to cure cancer once you've got it. But I also think you're going to modulate the incidence, like the mutational frequencies that are giving you cancer, right?
So if you think of cancer, in a way, it's easier to think about when it starts, because you say, well, it starts when you have a cell that has all the requisite mutations to be cell-backed uncontrolled growth. So if that's the point it starts, I think we're not going to affect that. But once that cell exists and now has to start growing and also saving the immune system, I do think that's probably what you're going to affect. In other diseases, like cardiovascular disease, where you can imagine things like autophagy could be quite modulatory, I think you can imagine that you're also even affecting the incidence, the exact point at which you'd say, okay, this is an atherosclerotic plaque or not.
What do we know about rapamycin and TOR in the brain, especially with respect to neurodegeneration? Yeah, that's a really interesting one, and that probably is a really important question for the future. So we know autophagy matters a lot in the brain. If you delete autophagy, and really, Mintushima was the person who kind of made autophagy.
interesting to lots of people. He was awarded the Nobel Prize. No, no, he wasn't. He was awarded the Nobel Prize.
He didn't share. He didn't know. Which I think was a bit of an oversight in my view. But he basically studied autophagy in the brain, made mutations, showed you got neurodegeneration.
So that was a really important finding. Connects up to lysosomal storage diseases, which, you know, autophagy, basically autophagy is on fusible lysosomes when you have that connection. So I think, like in all tissues, it's a bit of a double-edged sword. You clearly need M2O activity to maintain healthy synapses, certainly during brain growth.
If you make mutations around a growing animal, you basically don't have cortex. On the other hand, you clearly need to be able to modulate M2O1 to have some level of autophagy to keep the system healthy. Now, you could debate, is that in neurons? Is that in glia?
It's probably in both. People have made it in certainly neurons or suggested both, but then some of those promoters are a little bit dirty. But the real question in the brain is what modulates M2O1? Because it's not probably nutrients.
Because they're so constant, you mean? Exactly. Your brain, your body. Your brain prioritizes nutrients in the brain over it.
It basically protects. So if you take an animal and you fast it for two days, a mouse, it loses a lot of weight, 25% of its weight. And now you take every single tissue and you weigh it, every tissue has shrunk. Some like the thymus has shrunk ridiculously.
The kidney shrinks, which you wouldn't expect. The heart shrinks. The brain, nothing. Now, clearly, probably if you, in a mouse, you can't do that extremely fast.
And so the body protects the brain from a nutrient point of view. Yeah, M2O1 activity is high there. Clearly, we know that we have to modulate autophagy. So something must be inhibiting M2O1.
This is my peripheral argument for why, and I'm in a huge minority here, I do not think the brain is really the appetitive center. I think it's the modulator, but I, for that exact reason, think it wouldn't make sense for evolution to put our appetite center in our brain. It should be in the periphery. It should be in the liver, I think.
Yeah, but people argue that things that hypothalamus are in the periphery, right? Because they're not protected. There are parts of your brain like the hypothalamus. And the point is that it has to be, your appetite center needs to be regulated to something that senses very rapidly the pain.
Yeah, for sure. Yeah, for sure. And exactly where it is. And the bottom line is probably...