The Peter Attia Drive - #114 - Eileen White, Ph.D.: Autophagy, fasting, and promising new cancer therapies

Episode Date: June 8, 2020

In this episode, Eileen White, Chief Scientific Officer at the Rutgers Cancer Institute, describes the fundamental role of autophagy in the maintenance of health and prevention of neurodegeneration, cancer, and other diseases. She also goes into detail about the paradoxical finding that autophagy may benefit an existing cancer cell and help it to survive—a discovery leading to new possibilities in cancer therapy. We also discuss fasting (and molecules that induce autophagy) and the critical need to decode the proper fasting “dose” in order to improve human health. We discuss:   Eileen’s discovery that a specific oncogene blocks apoptosis [3:40]; Defining apoptosis and its role in cancer prevention [10:00]; How cancer cells use the autophagy pathway to survive [17:20]; Stressors that induce autophagy [29:15]; The importance of autophagy in the brain and liver [32:45]; The mechanisms that can trigger autophagy to support longevity [40:00]; Evidence for cancer treatment by blocking autophagy [42:30]; Types of cancer that are most autophagy-dependent [46:45]; The autophagy paradox [52:40]; Finding a molecular signal for autophagy [59:15]; Current knowledge gaps around fasting as a tool for longevity [1:13:00]; Rapamycin, metformin, and other molecules that may induce autophagy [1:22:15]; How to study fasting and exercise as longevity tools [1:32:50]; The Nobel Prize for autophagy research [1:36:45]; Eileen’s future areas of research [1:38:25]; A fasting strategy for Alzheimer’s Disease prevention[1:49:25]; Future study of metabolism and autophagy [1:51:30]; and More.  Learn more: https://peterattiamd.com/ Show notes page for this episode: https://peterattiamd.com/eileenwhite  Subscribe to receive exclusive subscriber-only content: https://peterattiamd.com/subscribe/ Sign up to receive Peter's email newsletter: https://peterattiamd.com/newsletter/ Connect with Peter on Facebook | Twitter | Instagram.

Hey everyone, welcome to the Drive Podcast. I'm your host, Peter Atia. This podcast, my website, and my weekly newsletter, all focus on the goal of translating the science of longevity into something accessible for everyone. Our goal is to provide the best content in health and wellness, full stop, and we've assembled a great team of analysts to make this happen. If you enjoy this podcast, we've created a membership program that brings you far more in-depth content if you want to take your knowledge of this space to the next level. At the end of this episode, I'll explain what those benefits are, or if you want to learn

more now, head over to peteratia MD dot com forward slash subscribe. Now, without further delay, here's today's episode. I guess this week is Professor Eileen White. Eileen is the deputy director and chief scientific officer, along with the associate director for basic research and the co-leader of the cancer metabolism and growth research program at Rutgers University Cancer Institute in New Jersey. She received her bachelor's degree from RPI and her PhD from SUNY and Stony Brook and her postdoc with Bruce Stoemen at Cold Spring Harbor Laboratory. Eileen's early work focused on apoptosis, but it was doing some of the work there that she stumbled upon atophagy. And that

is the focus of our discussion today. Now, if you're even remotely familiar with this podcast, you'll certainly know that the concept of autophagy has come up on so many previous episodes. It is a fundamental pillar of health and maintenance of health. We talk a lot about it in the context of fasting in particular. I have wanted to sit down with Eileen for a really long time and I don't think this conversation Disappoints, although we certainly could have gone longer. In this discussion, we talk about Eileen's career and how it morphed from studying Apeptosis into a topology. We go into describing the regulation of a topology both

Metabolically and otherwise. And then we spend a lot of time talking about the role atophagy plays in both the prevention of disease and also the treatment of disease. And I think this is where it gets really interesting, especially around cancer. And I think that that's potentially one of the most confusing aspects of the entire discussion on atophagy. And that's actually one of the reasons I really wanted to talk to Eileen was to better understand something that at the surface seems confusing to me, which is that autophagy seems to very clearly protect a person or an organism from getting cancer. Yet once someone has cancer, it appears that autophagy may disproportionately benefit

the cancer cell versus the non-cancer cell. So we tease this idea apart, along with talking about the amazing work that her lab has done to demonstrate the importance of autophagy in preventing Alzheimer's disease and neurodegeneration, along with the benefits of metabolic health. And of course, we do talk about the age-old question that many of you have heard me go on and on about, which is, how do we delineate and understand the dosing and frequency of fasting as a tool? In other words, when I talk about doing a fast of three days every month versus seven days a quarter versus five days a quarter, how could we possibly get a handle on what the ideal strategy is? And so we talk a lot about that as well. And I'm actually quite hopeful that from this discussion

comes some research that can shed light on that. So without further delay, I hope you enjoy my discussion on autophagy with Eileen White. [♪ OUTRO MUSIC PLAYING [♪ Eileen, thank you so much for extending your trip in San Diego for a day to come and make time to talk with me about this stuff today. Oh, it's my pleasure.

I'm looking forward to it. I don't know if you remember this, by the way, but David Sabatini introduced us a few years ago. Do you remember? I do. Yeah, I still have my notes from that phone call five years ago. I took about 20, maybe not 20, that's exaggeration, maybe 10 pages of notes in my journal and probably have gone back to those a dozen times in the last five or six years.

So I was appreciated when people just pick up the phone and talk to total strangers for no reason. So that's great. Well, you were excited about the science and so nerds like me like to talk about science. Well, let's actually start from there. Tell me where your interest in science came from. Was it something that was always in you from a young age where you just naturally curious?

Yeah, I've been asked that question many times before. I come from a family where there was an interest in science. My mother was an elementary school teacher and my father was a lawyer, but he always wanted to be in science. And all of our discussions were related a lot to new scientific discovery. So from an early age, I was introduced to science, which was probably unusual. And I then went to college and majored in science and biology and continued from there. And I decided when I was an undergraduate,

I wanted to get a PhD in biology. And I was very fortunate to go into graduate school in the department led by Dr. Arnie Levine who discovered P53 and that was an inspirational experience because he has got scientific insight that's absolutely incredible. And then I went on to Quiltzpen Harbor Lab where I was a post-doc with Bruce Stillman and again another incredible scientist and it was an incredible scientific environment. There were a whole cadre of investigators there that were making major contributions in the

field of cancer at the time. And it was just a very thrilling experience to be in an environment where once a week there was some fabulous discovery and everyone was excited about it. There were even, I think, two Nobel prizes awarded while I was there. And I joined the faculty at Colesbury Harbor after that and then I moved on to Rutgers.

And I had the fortunate experience of building a cancer center. So when I went to Rutgers to be on the faculty, there was no cancer center. But shortly after I arrived, they hired a cancer center director Bill Height from Yale. And I joined him to help build what's now the Rutgers Cancer Center or the Rutgers Cancer Institute,

which went from nothing to now there are multiple buildings, there are 11 hospitals in our health system. We have 240 something members of the Rutgers Cancer Institute and we have, we're a consortium Cancer Center with Princeton University and so it was very thrilling for me to not only have maintained my scientific interest by running a research lab, but also help expand and grow something from nothing where now we're treating large numbers of patients

in standard of care and clinical trials and making large discoveries and moving cancer treatment, advancing cancer treatment as fast as we can. And how many years have you been at Rutgers? I joined there in 1990, so I've been there for a long time, but I moved about 12 years ago. I moved from one research building to be physically in the cancer center where I could be more directly helpful. So where in your journey did atophagy peak your curiosity? I was purely serendipity. So this

goes back to when I was a post-doc with Bruce Still at Coltspren Harbor, I was given an oncogene to study. That time, they had just sequenced the adenovirus genome. They knew what genes caused cancer in the virus. I was given one of those genes and said, figure out what it does. That was a dream project for a postdoc. What I found was that this gene was a viral homologue of BCL-2. BCL-2 is a gene, it's a human oncogene, and it functions by blocking apatosis or program cell death.

And so that was transformative to me, and took other people a while to realize the importance of that, that one novel function of cancer is to evade cell death. That field grew. We and others contributed cloning the other genes that regulated apoptosis. We figured out how it all, the mechanism by which it worked, and the pharmaceutical industry started developing inhibitors of BCL2 to promote apatosis and cancer. And that was the ultimate goal, was to make tumor cells die and have a drug that will do that.

And once that happened, the field of apatosis, I think, sort of, we accomplished what we wanted to accomplish. We understood everything and that led to the development of the first of many drugs that were in clinical trials. And in fact, my lab is still involved with taking those drugs and putting them in patients in optimizing their use in solid tumors. So while the field of apoptosis matured to the point where things were being translated, we made a

serendipitous discovery. We had engineered tumor cells to be unable to undergo apoptosis. They were refractory to being able to commit suicide. Can we pause for a second there, Alene? And just let's explain to folks exactly how apoptosis works because shortly, we're going to obviously contrast this with a topogy. They have common threads, but they're different. So let's go down the path of what does it take

to get a cell to undergo programmed suicide? So there's a family of proteins called the BCL2 family. They come in different flavors. There are the BCL2-Lac proteins which inhibit apoptosis, so they keep tumor cells alive. And BCL2 is the prototypical member of that family and is up-regulated and amplified and translocated in many cancers to do exactly that. And are antagonizers of BCL-2 and it's related proteins, these are called the BH3-only proteins

and they are often activated to inhibit BCL-2, to trigger apatosis. And then there's the core apatotic machinery that triggers apatosis, and this is back and back. They reside in the mitochondrial membrane, and when they're triggered to undergo apoptosis, they eligomerize and poke holes in the mitochondrial outer membrane that releases proteins that activate proteases to grade the cell. And BCO2 and BCOXL, all the anti-apoptotic proteins are involved in antagonizing this process.

And what are some of the things that would have to be going wrong in a cell for it to go down that suicidal pathway? So for example, mitochondrial injury that is irreversible, genetic mutation that is unfixable, like what are the suite of things that basically take a cell down the path of, I can't fix this, and being around here and replicating is going to be dangerous to the host, I got to take myself out of the game. Right. So mitochondrial damage, and certainly trigger apoptosis, but probably the best way to explain

to disease by using the example of P53. So P53 is a tumor suppressor and a transcription factor. And some of the transcriptional targets of P53 are proteins like Puma and Noxah, which are these antagonizers of VCO2 and activators of back to back. P53 is a tumor suppressor. and activators of BACS and BACK. P53 is a tumor suppressor. One of the functions is to promote apatosis

to prevent an emerging cancer cell from progressing. One mechanism by which P53 does that is by turning on the transcription of Puma and Naxa, and then that will antagonize BCO2 and initiate apoptosis. So then the question becomes what activates P53 to do that, and that could be a long list of things from DNA damage, from oxidative stress, and so forth. So you can think of something bad happens to an emerging cancer cell, and then P53 gets activated. And one of the tumor suppression functions of P53

is to turn on these promoters of apoptosis that antagonize BCO2. Now, loss of function in P53 probably accounts for half of all cancers, correct? Right. And I assume that you have to lose both copies of it or is losing one copy sufficient. Well, what happens in most of the time is not deletion of P5D3, but rather a point mutation that reduces function.

It's more of a dominant negative. So in fact, there's even evidence that there's a gain of function. So there are hotspot mutations in P53 that are very common in cancer, and P53 functions as a heterodimer. And what these mutant P53s do is that they end up entering into a dimer with wild-type subunits, and that interferes with the function of the complex. So yes, in that respect, it can be a loss of function of the P53 heteroid dimer, but there's evidence that it not only causes a loss of function, but it actually may do other things as well that are cancer promoting.

That's just a great example of the nuance of evolution, right? I mean, in med school, the classic teaching, you know, one hundred years ago for me was P53, loss of function, oncogene, gain of function, black and white, of course, it's never black and white. Right. So what is the wreckage of apoptosis? So when a cell undergoes apoptosis,

to everything outside the cell, inclusive of the immune system, what becomes visible. In other words, does an apoptotic cell, once it dies, elicit any immune response, or does the process of apoptosis yield sort of an inert body of cellular matter that just goes away? I'm not so sure. I'm the best person to answer that question. I think the whole idea initially was during the process of apoptosis you would get protein degradation and packaging of pieces of

the dead cells into these apoptotic bodies. And then that would reduce inflammation. And then there's evidence that macrophages can then go and take up these apatotic bodies, and that may facilitate antigen presentation and so forth. So it's possible, for example, that if you have a cell that has become cancerous, either through a gain of function, loss of function, but whatever, there's some mutation that now renders this cell to go down a pathway of cancer. It fortunately, it undergoes the apoptotic transition. The macrophages take it. Is it likely that you get an immune response to that that is protective in the long run against similar mutations? Because, I mean, even though the macrophage is part of the innate

immune system, does that ever translate to the adaptive immune system such that you gain some long-term immunity from that specific type of mutation? Yes, I think something like that occurs, and I'm just thinking of the wrong person to answer that question. I could give you the better names of people that can do a better job, but the, I think the best way to compare it is to contrast it with the chronic cell death. So in apoptosis you have proteolytic degradation and of a cell and packaging it into these bodies and you say, well, does that limit information? Well, the way to explain how it does is to compare it to a different form of cell death like the crecrosis. So necrosis is cells' lice, and that is very pro-inflammatory. You have nucleic acids released.

You have essentially everything is released. Including mitochondrial content, which is probably the most immunogenic given its bacterial origin of the DNA. Absolutely. And so... Apoptosis, what I'm hearing you say, is apoptosis is much cleaner than ne the DNA. Absolutely. And so... A poptosis, what I'm hearing you say is, A poptosis is much cleaner than necrosis. Absolutely.

So now let's talk about autophagy. Let's contrast the autophagy with apoptosis. That's right. Well, before we get to that, I should go back to your original question of, like, how did we start working on autophagy? And this sort of bridges us to what you just mentioned. So, when we disable apoptosis in a cancer cell, it can't commit suicide.

And we're doing that all the time. And we could show that then the tumor cells become more tumorogenic. But what we didn't expect was the extraordinary propensity for survival. We could leave the cells out and put them in buffer. They wouldn't die under extraordinary circumstances that we couldn't explain. So why would a cell that just couldn't commit suicide survive in buffer with no nutrients at all? And just for context, this is the mid-80s, late 80s.

This is the mid-80s. No, it's probably later than that. It was probably early 1990s. And so it was a conundrum. I mean, just because a cell can't commit suicide doesn't explain how it can be a cancer cell can just sit in buffer and be fine. And we puzzled over this, like how can this be? And then we discovered that what these cells had done was turned on autophagy and we're

using that for survival. Before you go down that path, help me understand something. What did you observe about those cells that were sitting there in the absence of nutrient surviving? Did you notice any metabolic changes that were unusual? Like, what was your clue that they were able to usurp the environment they were in? Inactive desperation, we tried a bunch of things and nothing was informative. And then I told the people in the lab, why don't we just look at these cancer cells onto the electron microscope? And that way we can see everything, because we couldn't understand how they could be surviving above her.

And when we got the electron micrographs back, we saw something we had never seen before, and all these double membrane vesicles all over the cell. And so that when we finally fell, all those are out of agasomes, which we had never ever seen. But wait, how did you, I mean, first of all, this is just to me one of the beautiful moments in science that I think it's so important

for people who don't do science for a living to understand that while science is 99% failure, every once in a while you have a moment like that. Are you a reek of moment? Yeah, it probably makes up for 10 years of failure. Yes. When you realize in that moment you are seeing something that has never been seen before and therefore this is the cusp of new knowledge. That's right.

That's happened to me a bunch of times in my career, which is fortunate. And this was one of those moments. And then we started to read, well, what are these autofaggisomes? What do they do? And then we realized from when we looked in the yeast literature, they were meant to capture intercellular proteins in organelles and bring them to the vaguole of the mammalian lysosome for degradation and recycling, and that this was a mechanism by which yeast survived starvation.

And what was the tumor line or what was the cell line you were doing this in? At the time we were using kidney cancer cell lines. Mice or human. These were mouse. But it was basically the first time this had been seen in mammalian cell line? No, I think people had seen autofagosomes before. I mean, you got to remember in the olden days when electron microscopes were first available, that's one of the things that people did was describe all kinds of different

processes. So what are phagosomes were known to exist, but there was very, very little information, almost no information on autophagy and cancer at the time. So we went into this area where there was almost no information. And so the first question we asked is, okay, well, the yeast data tells us that when you see auto-faggisomes, that means cells are starved and they're recycling and they're using this to survive. And we hypothesized that that's what was going on in these cancer cells.

And if that was the case, if a topogy was a survival pathway in cancer, that was a game changer. We had to understand it, and we had to demonstrate that that's what was actually happening. And then if that was the case, if cancer cells had usurped the autophagy pathway for their survival, then we needed to inhibit autophagy for cancer therapy. The first thing we did was we looked to see what would happen if we inhibited autophagy in these cancer cells. And the answer was very simple.

In many, many circumstances, in many different cell lines that we looked at, when you inhibited autophagy, the survival of the cancer cells was reduced. Let me interject for a second and ask a question. I don't know if you ever did this experiment, but if you took the kidney line and the kidney cancer line, so basically the same histology from the same tissue,

from the same animal, but one has the oncogenic properties and one does not. And you put them in the identical nutrient deprived stress. Can you quantify the amount of autophagy or the efficiency with which those two cells undergo autophagy? In other words, is cancer simply preserving the atophagy capacity that it had as a noncancer cell, but not enhancing it or not having any attenuation of it? Is it simply just, hey, this just happens to be something that gets preserved as you go from noncancer to cancer? Or is there some qualitative or quantitative change in the character of autophagy as a cell mutates? That's interesting.

So let me see if I can unpack that. So if you have the general observation is that normal cells in the fed state don't have autophagy on it, functions as at a very, very low level. And if you starve cells or mammals for nutrients, then there's a massive upregulation of autophagy. What was striking about the cancer setting was that even in the fed state autophagy was elevated. Oh, so there is a fundamental difference there. Yes. topology was elevated. So there is a fundamental difference there. Yes.

And then if you stress them, it goes up even further, but the problem is is that when it's already high, how much higher can it go? And just to be clear, Eileen, this is in vitro, so you can't even make, when you said that, the first thought that came to my mind was, well, maybe the reason is they're undergoing a different stress, which is, for example, a vascular stress, a hypoxic stress, because, you know, heaven got enough Vaget, or they haven't created enough, in other words, the apoptosis is going up despite being fed because there's something else that's impairing them. But if what you just said is true in vitro, then that wouldn't explain that, would it? In other words, if they're not limited for oxygen, if they're

in a petri dish and this is happening, my hypothesis wouldn't make sense. That would only make sense if what you said was true in vivo. Right. So in the fed state, the cancer cells already have elevated autophagic flux. And when you fast them, it does go up, but it only so high it can go. I see. So this probably, this probably, it's really funny. I'm sure you familiar with the paper that Matt Van der Heiden and Lou Cantlin Crack Thompson wrote in Science in 2009, which was at least to my knowledge the first time that someone offered an alternative explanation for the Warburg hypothesis, which is, hey,

it might not be that the mitochondria of the cancer cells are defective and can't undergo oxidative phosphorylation. It might be that they're optimizing for growth as opposed to metabolism. They don't care as much about ATP as they care about building blocks, and therefore they're deliberately taking an inefficient route of glycolysis to lactate because they want the cellular building blocks. And that might be the explanation here, is that the tumor cell is undergoing more constant proliferation,

and therefore they want more building block. Exactly right. And in fact, when we look at cancer cells and we study their metabolism, what we've noticed, and this is something that we found, and it's been a common observation, is that nucleotides seem to be rate limiting. And so the metabolism of a cancer cell is designed

to facilitate denobosynthesis of nucleotides. So, that's really interesting, isn't it? When you're really something about, like, you think of all the things that could potentially be rate limiting to a cell. Think of how many phospholipids, for example, they need to build all of those cell membranes. And yet it's the nucleic acid to continue to propagate its DNA that becomes rate limiting. That to me is very interesting. I wouldn't necessarily have ever guessed that. We may learn more as going forward, but that is what seems to be a recurring theme. But it's not just DNA, it's also RNA, and you have to remember that RNA and ribosomes make up a huge amount. They're a much greater demand. Exactly. I think David Sabetini has mentioned this many times

that a large amount of the mass of a cell is ribosome larynae. And he had a beautiful paper where he was making the argument that ribophagy, the autophagy of ribosomes, was an important metabolic survival mechanism. And you could think of ribosomes as being a depot, a storage depot for not only nucleic, look, acids, but also protein, and when a cell is stressed or starved, it doesn't need to make protein, so it doesn't really need large numbers of ribosomes. And so the autophagy pathway can cannibalize those ribosomes because they're unnecessary. And then recycle all that protein and nucleic acids to support survival. Mammunika, you mentioned, of course, that some of this had already been observed in yeast.

And the moment we start talking about things that are true in yeast and then true in animals, mammals, for example, are higher or animals. We're talking about a billion years of evolution here. So this ranks as one of the few things that seems remarkably conserved over evolution. As a general rule, that makes it very important. Do we have a sense of when this first showed up? Again, I might be out of my league. I mean, I know it's certainly a big function in yeast.

Prior to yeast, I don't know. Yeah, but it's amazing. I mean, it's sort of, it's in the category of mTOR. Yes. Something that is so important that it just doesn't really seem to change over about a billion years. Rule of thumb, it matters. That's right. And I think when you compare how yeast does autophagy and how mammals do autophagy and what they're using it for, it just looks like mammalian version of autophagy is a little bit more complicated. It's probably, they have probably more different circumstances where autophagy might be necessary,

but the basic process is surprisingly the same. What are some of the other stresses that induce atophagy? And let's maybe just for the moment even start with just in a normal cell. So let me sort of re-synthesize what we've talked about. Clearly nutrient deprivation is one of the biggest triggers for atophagy. And I mean, maybe just for the sake of time, I'll kind of throw this out there and you can correct me if I'm wrong. But I've always sort of thought of this through three pathways at the sort of mechanistic level.

So you have sort of the mTOR pathway, which is mostly sensing amino acids. You have the AMPK pathway, which is mostly sensing energy and ATP in general. And then you have sort of the acetylcoA protein deacidilation pathway, which is also just basically sensing substrate of fatty acid and glucose. Is that sort of a fair way to say that those are three ways that low nutrients can still

trigger the same pathway? Yeah, and I think you could add on to that stresses that result in organelle damage, such as depolarization of mitochondria or dysfunction of mitochondria, activation of protein misfolding and generation of protein aggregates. So I think there's the things that are directly related to metabolic signaling that you've mentioned, but then there are other stresses that also can tie into the autophagy pathway. Are there other stresses like the nutrient that are stresses that come from outside the cell to inside the cell?

So the protein misfolding, the mitochondrial depolarization, those are things that are occurring as damage within the cell that stress. Do we know anything, for example, about temperature? Do heat shock proteins stimulate autophagy in extremes of temperature? Does exercise, I mean which obviously is in the short term quite stressful, how potent is that at inducing etothogen in a normal cell? So temperature wise I would fully expect that temperature extremes would induce protein misfolding and induce etothogen as a remedy for that, but I don't recall any studies on that.

In terms of exercise, that's very well studied that exercise induces autophagy very potently, and you actually need autophagy to, because exercise damages the muscle, and autophagy is one of the processes that helps mitigate the damage that occurs during exercise. What about hypoxia? Oh, potently. Hypoxia potently induces autophagy. Wow. And in fact, one of the first things we did was when we looked at tumors, tumors are well known to have hypoxia in the center when we engineered tumor cells to be genetically deficient for autophagy, and you look at them, they're completely hollow. I mean, they may have no organelles, nothing.

Not the cells are hollow, the tumor is hollow. Oh, wow. The further the cells get from a blood supply, meaning the more susceptible they are to hypoxia, they're dead. Right. So if you take a tumor, if the middle is hypoxic, that's where your atopology is most active. And if you genetically ablate autophagy, and the tumor end up with the hollow tumor, because the tumor cells in the middle don't survive.

Yeah. So it does come back to this idea that we talked about earlier about hypoxia being potentially one of the things that autophagy is protecting cancer from. Absolutely. How easy is it to create an animal model that is unable to undergo autophagy? How difficult is that from a knockout perspective?

Well, it's been done and we've done it, and it can be done different ways. So the original malstrains that were made were deficient in either of two of essential atopology genes, one called HG-5 and another one called HG-7. And these mice were developed in Japan and these mice are born but they failed to survive the neonatal starvation period. So when mammals are born, meaning they fail to survive the neonatal starvation period. So when mammals are born... Meaning once they are cut off from an umbilical nutrient source, it's almost like they have

a glycogen storage disease. You know those conditions where you can't produce any glycogen, it's uniformly fatal if not treated the moment you're cut off from an umbilical source of nutrient. Right, so that neonatal starvation period between the cutoff of the pacenta and suckling is a common feature in mammals and there's potent induction of autophagy during that period. I can't speak for humans, but certainly I would think so. And in mice, that's exactly what happens. And so these autophagy-deficient newborn mice don't survive. Wow.

That really, I mean, again, I think that simply underscores the evolutionary preservation of something. If you knock it out, it is uniformly fatal. Do not pass go. Do not collect $200. You're gone. And then what we did was, yes, a different question. It was like, what happens in an adult mouse? So in the newborn mouse, it's very different situation

because any newborn mammal, they don't have any fat. They have no reserves. And so when the Japanese group did the extraordinary thing of trying to force feed these autophagy deficient newborn animals, and they didn't extend their survival very much. Now, is this Yoshenari's group? This was the Boromizashima and the Kumatsu group, I believe. I think it was coming from those two labs.

So what happened in that experiment? They discovered that the mice died shortly after birth, and then they realized that, well, they suspected they had a metabolic problem, and they weren't succulent because they were probably too ill to, by the time they would have been able to, so they forced fed them, and that allowed them to live for 24 hours, but they still died anyway. And then are you able to induce an autophagy knockout in an adult? Yes, so that's what we did because we realized that the newborn animal is... It's just too fragile.

Too fragile, they have no nutrient reserves. And actually in the setting of cancer, we're thinking of you want to treat an adult with a tumor so what's happening in autophagy and a newborn animal isn't even relevant. And so we engineered mice where we can take an adult mouse and give the mice a chemical so that an essential autophagy gene will be deleted throughout the entire animal. So one day, they're in a don't-mouse with autophagy, and then a few days later, they're in a don't-mouse with no autophagy. And these mice were very extraordinary. They lived for two to three months, and then they died predominantly of neurodegeneration. Autophagy is very important in the brain over

the long term. But if we fasted the mice, they were all dead within 16 hours. Let's unpack that again. That's pretty remarkable. So you take a normal mouse that's got through the vulnerability period of infancy and you genetically knock out its capacity for autophagy. The first thing you observe is if you fast it for 16 hours, which admittedly is a pretty long fast for a mouse that might be the equivalent of fasting a human for a week, but that degree

of nutrient deprivation is uniformly fatal. If you continue to feed them well, they only survive another couple of months because they ultimately succumb to neurodegeneration suggesting that the role of atophagy in preventing neurodegeneration is essential. And it's really not surprising when you think about the role, everything you talked about with protein misfolding, and when you start to think about the toxicity that are driving neurodegeneration using Alzheimer's disease specifically as an example, there's a lot of crap that's basically getting accumulated in neurons. This would be an elegant way to suggest that

atophagy is keeping that at bay. Exactly right. So one other way of looking at it is what tissues are more atophagy dependent than others. Exactly. Brain would be really important and there are a few others. And what we've noticed when we looked at the mouse, the lactotophagy, when we genetically deleted the autophagy gene in the adult mouse, was that there were tissues like the brain that were very sensitive, and there were other tissues like the lungs that didn't have any phenotype. So wait, in other words, when those animals ultimately die of nerve generation and you undergo the pathology analysis, obviously the brain is where you see the cause of death.

You're saying in the lung, it looked completely normal. Relatively normal. What about liver? Liver was very sensitive. So it doesn't lead to the death of the mouse. So if you did a liver specific knockout of an essential autophagygy, those might have teotosis and their liver gets huge and whatever, but it doesn't kill them. I mean, they can live for quite a long time. So you induce fatty liver disease. So again, suggesting that autophagy probably plays a role in preventing fat accumulation and live for quite a long time. So you induce fatty liver disease? Yes. So again, suggesting that autophagy probably plays a role in preventing fat accumulation in the liver. Exactly right.

And also protein aggregate formation. One of the other phenotypes of stiotosis is the accumulation of these malere bodies, which are large protein aggregates composed of a protein called P62. When you lose autophagy and the liver, you're causing accumulation of fat, accumulation of protein aggregates, but the liver manages to tolerate it. The brain, however, is a different story. And if you have post-mitotic neurons where they don't have the capacity to do that, I mean, when they accumulate the crap, as you said, then it's game over. mytotic neurons where they don't have the capacity to do that. I mean, when they accumulate the crap, as you said, then it's game over.

Was there evidence that the brain in some last ditched effort to survive was undergoing more apoptosis of neurons? Yes. That's the common feature of these animals is increased apoptosis in the brain. But before that, you see all kinds of terrible things going wrong. This has been part of a major effort to generate autophagy stimulators as a remedy or as a means to delay neurodegenerative diseases. I want to come back to this later on in the discussion, but I'll just plant the seed now.

Obviously, fasting is one of the most potent stimulators of autophagy. I spend a lot of time thinking about how does fasting fit into our toolkit of longevity. A big part of longevity, in fact, probably the single most important piece of longevity when it comes to the lifespan aspect of it. So, you know, you think of lifespan versus health span, how long you live versus how well you live. On the how long you live front, I think it's very safe to say, based on all of the animal data, and frankly, all of the centenarian data, that the key to living longer is delaying the onset of chronic disease. So, even when you look at centenarians who are genetically gifted

with tools to live longer, if you unpack what the gift is, it's delaying the onset of the disease, not living longer once you have the disease. So the centenarians, once they get cancer and once they get heart disease, they die at about the same rate over the same duration as the rest of us schmucks. The difference is they get those diseases 20 to 25 years later. And again, that suggests to me that if you want to live longer, you have to delay the onset of these things, not live longer once you have them. And so it's hard to think that fasting doesn't play an essential role in that. When you realize the role that fasting plays in the mitigation of Alzheimer's disease

and metabolic disease, of course what we're going to come back to in a second is cancer, which seems to be this conundrum. This is the needle we're going to want to thread a little later down the line. I'll plant the seed now, but I do want to come back to the idea of ways that we can also induce autophagy, sort of pharmacologically or chemically. The first thing that would jump to your mind is anything that mimics fasting. The first thing that comes to mind would be metformin, rapamycin, things like that, that what we talked about earlier, just for the listener to sort of tie this together, we talked about these huge pathways that tell the body nutrients or scares.

So when mTOR activity is down, that's a sign that we're deficient in amino acids, but we can also do that with rapamycin. When AMPK is up, that's the cell being told we're deficient in ATP. Another way you can do that is to give metformin. We haven't talked about sertuins yet. Maybe I'll pause for a moment.

Do we have any sense of what sertuin activity does in autophagy? I'm not familiar with that literature. Okay. I was going to say, because then you could get into the whole NAD versus NADH ratios and how that might factor into it. So again, I'm really curious about this through a clinical lens as well, which is what is these suite of products. Almost just saying that out loud, so between the two of us, we remember to come back to this. But I now kind of want to get back to your story, which is, we've got these mice, you've got this much more elegant experiment now,

which is you're actually going after the phenotype of interest, which is an adult in which you inhibit a topology. What was happening in that animal if it had cancer? So did you have the experiment where you had an adult with cancer, then you knock out a topology? That's actually one of the reasons we made that mouse. So we had two questions we wanted to answer. One was, if you inhibited autophagy and an adult mouse, what would happen? Because if they died in an hour, then targeting autophagy

for cancer therapy would be pointless. Especially if you can't do it specifically. That's right. So the answer was it didn't die in an hour. They died in two or three months, which was actually good news, because that meant that there was a potential window of opportunity for inhibiting autophagy for cancer therapy. And I'm sorry, Eileen, when they died in two to three months, was it still from the neurodegenerative disease?

Yes. And did they still have cancer at the time of death? We had to first make a mouse that lacked where we could switch off autophagy and find out what happened to that mouse. Okay. So we did that and we saw that they died of neurodegeneration two or three months, which was good. They died immediately. Then we would have stopped. There would be no point in

trying to make cancer in that animal. But we did learn that they were intolerant to fasting, which was perfectly consistent with everything we knew about what autophagy functionally did. So then we moved to the second step, was to do the experiment that you just suggested, step was to do the experiment that you just suggested to make cancer in that mouse. And then after the mouse had cancer, to then shut the autophagy pathway off. And then to ask the key question, which died first, the mouse of the tumor. And the answer was the tumor died first. Wow. Okay.

So then here's the gangster question. Once the tumor died, could you reactivate a topogy to prevent the neurodegeneration or is it a one switch direction? That required a different type of mouse model. So what we were doing was making a mouse with cancer and then once the mouse had lung cancer in this case,

we deleted an essential autophagy gene in the entire mouse tumor and all, but the gene was gone. So it wasn't like we could turn autophagy back on in that model. But since then, one of my trainees in collaboration with the lab in the UK, they have developed a model where they can toggle autophagy often then back on again. And what they've seen is a remarkable capacity of the normal tissues to restore themselves. So the experiment would be to have a mouse to induce an SH RNA to a specific autophagy gene to down-regulate the expression and inhibit autophagy that way, and then later on, take that SH RNA away or shut it off and restore normal autophagy in the mouse.

And you see a lot of capacity for the tissues to restore themselves back to normal. So that experiment basically becomes the proof point that says targeting atophagy and cancer makes sense. That's probably the most elegant description you could provide of that. That's right. I think it would be better if we had specific targeted therapies against some of the enzymes in the otopathy pathway because these are all genetic experiments and it's not

exactly the same right you might not get the complete penetration with a drug or inhibiting a protease is not exactly the same as deleting the gene. But this is all what's called proof or principle that the concept of inhibiting autophagies in cancer is valid. So what do we know today about what you've just described as it pertains to two things? So I want to slice the data across two variables. The first is tissue type or histology of cancer, and the second is underlying genetic mutation. So I know that a lot of what you're describing is clearly true in K-RAS mutation. What about other drivers? We and a number of other cancer labs that use genetically engineered mouse models for cancer have been banging

away at that for a number of years. And what we've learned is that K-RAS-driven, lone cancer and pancreatic cancer are extraordinarily autophony dependent, and you do it, you know, make the mouse models, and the tumors are very susceptible to the functional loss of autophagy. Can you briefly tell folks what a K-RAS-driven cancer does, like what is it about the mutation that drives the oncogenesis? So K-RAS is a GTP binding protein that is responsible for activating what's called the MAP kinase pathway. And this pathway is very key in driving cell proliferation.

And so cancers have a mutation in RAS or mutations in RAS that leave it in the GTP bound or on state. So there's perpetual growth signaling through the MAP kinase pathway. Which of course is the hallmark of cancer, which is its unresponsive to cell signaling, and when you are fixed in the on position, you can't turn off, and basically that is cancer. That's right. And what's particularly interesting about raster than cancers is that we have been very unsuccessful in drugging rast.

And there's recent hope that the system, the particular subset of the mutations in rats that involve a cysteine residue that there are now drugs that target that. There's hope after decades of failure. Is the primary issue in the failure to drug rats that you can't do it without creating toxicity for other cells that are non-cancer or that it has too many workarounds to whatever you put in place. I think too many workarounds is a common problem. What they've done is that they said okay targeting rast is difficult and let's go downstream

of rast. And try MAP kinase. Right, right. So there are inhibitors of raff, mech, and Erk, which are downstream of Raff, and those are actively in use in the clinic, but they seem to be not durably effective in Raff, driven cancers because of the workarounds. Yeah. So are there mutations

or mutant drivers of cancer that we know are not dependent on autophagy and unresponsive to the autophagy blockade? Yeah, it seems like there's a spectrum. So rash of incancers are particularly sensitive. B-Raff driven cancers, like B-Rap B600E is a common B-Rap oncogenic mutation and those cancers are particularly sensitive and those the ones that were examined were lung cancers and melanoma. The B-Rap B600E mutation in melanoma is very common homologous recombination defic deficient breast cancers, and those would be, well, I mean, those would be models of hereditary breast cancer. Those are very sensitive to loss of autophagy. APC deficient colon cancer is another example.

What about non-APC driven colon cancer, which of course is the majority of it? What do we know about that? I don't think I can remember seeing a paper. I remember the APC deficient model. That's, in fact, the most commonly used model. Anything we know about prostate cancer or other hormone sensitive breast cancers? So prostate cancer is sensitive. We did that work. And hormone sensitive breast

cancer, I'm not recalling right now, but there are a long list of cancers that are sensitive. The sensitivity is not all equal. For example, B-raft driven cancers are very sensitive. More so than rastroven lung cancers, you just compare lung B-raft lung cancer to r to lung cancer, the B-raft mutant lung cancer is more sensitive. The most sensitive cancer that we've encountered is in fact, Ras-driven lung cancer with LKB mutations. This makes a lot of sense, too. One of my trainees who has

their own research lab hypothesized that we sat down and we thought what cancer would be, would you predict to be most autophagy dependent? And it should be a cancer with loss of LKB1. LKB1 is a tumor suppressor gene that's involved in activating AMP kinase. And AMP kinase activates autophagy as a survival mechanism to low energy. And so there were a whole class of lung cancers that have lost LKB1.

And as a result, they can't activate this protective mechanism. This is Dr. Jesse Guel, she made this mouse model, rash of a lung cancer without LKB1, and low and behold, when you delete an essential autophagy gene, you abrogate tumor genesis. So it makes a huge amount of sense. LKB deficient rash of a lung cancer is probably the number one sensitive tumor. So how do we reconcile these two observations that almost seem to have a difficulty co-existing? So the first is everything you've just stated,

which is pretty clear and unambiguously suggesting that autophagy is at least for a number of cancers an important part of their survival and proliferation. And we contrast that with an abundant body of literature that suggests that when you combine fasting, which is a potent inducer of autophagy with chemotherapy, for example, you enhance its efficacy. And we can speculate about why that might be the case. These two things, although not directly comparable,

seem a little bit at odds. How do you think about those things? I would probably think about it in a slightly different way. So if you want to get at the two different roles of autophagy, one is cancer cells, you're surfing it and turning it on for their own survival. Then the other side of it is when we know that autophagy is protective. We know what happens if you have a mouse without autophagy. Many terrible things happen. It takes a while, but the mice die of neurodegeneration.

Can I interrupt for one second? I'm sorry to do this, but I just forget this question. I want to come right back to your thought. In those animals that died of neurodegener disease after two to three months, did they show an increase in tumor genesis in any other tissue? No, they don't. But if you make a mouse where you bypass a neurodegeneration by knocking out an essential autophagy everywhere else,

okay, but not the brain. Then those mice will get benign hepatomas, so benign tumors of the liver, but that makes sense too. Think about autophagy in normal tissues. We know it's important because if you knock autophagy at an amouse there's tissue specific but gradual deterioration ultimately leading to neurodegeneration and you end up with stiotosis, fatty liver disease. The brain phenotype can be explained as we discussed before, neurons and the brain need this protein and organelle quality control function. They're post-mitotic, they have to have a way of getting rid of the garbage. In the liver, what happens when you damage the liver?

It regenerates. It's got infinite capacity. That's right. But what happens when you... Unless there's too much inflammation. Exactly. So you end up with when you inactivate autophagy and liver, you end up with these chronic cycles of damage, repair, and chronic inflammation. You end up with when you inactivate autophagy and liver, you end up with these chronic cycles

of damage, repair, and chronic inflammation. And that is oncogenic. And that's not, you know, it's particularly obvious in the liver. The pancreas as well. Very, very sensitive to that inflammation. So I think what this is telling us is something very important. It's telling us that a main function of autogy in tissue homeostasis is to preserve cellular function to be normal to prevent chronic damage and inflammation and tissues that are susceptible

to cancer as a result of chronic damage and inflammation. Autophagy is highly protective. Which again, think about how complicated this is. Now I'll bring us back to the question I posed a moment ago, but using this example. Why does an affle de ultimately lead to cancer? Because if you have enough accumulation of fat, you get enough inflammation,

you're going to get a petticellular carcinoma, saying with pancreatic cancer. This is why alcohol is such a horrible molecule. So toxic to the pancreas, to the liver, and you sow those seeds of inflammation and low and behold, you're increasing this risk of cancer. So on the one hand, we know that autophagy helps ameliorate that.

It cleans that up. It buffers that. At the same time, we just realized a moment ago, oh boy, once you do get pancreatic cancer, it's a K-Rastrov and cancer, a topogy is helping it. That's right. Now, let's come back to the question I posed a moment ago that I so rudely interrupted you and answering, which was, how do you reconcile these?

I think it's a matter of thinking of the role of a topogy in cancer as being context dependent. On the one hand, functional autophagy can delay the onset of chronic damage and inflammation that are known causes of cancer in particular tissues such as the pancreas and the liver amongst a few others. So I think that stimulating autophagy through fasting or through pharmacologic means at one point can be thought of as preserving health. But once you have a cancer,

I think it's a different ball game. And at that point, it's a completely different context. And in that setting, what we've learned as inhibiting autophhology is preferentially damaging to the tumor compared to the normal tissues. And then going back to the other literature, which looks at the efficacy of fasting combined with chemotherapy, which is superior to just chemotherapy, do you think that the reason for that is that the chemotherapy itself, maybe once you're rendering the cells

more sensitive to chemotherapy and also potentially generating a more durable immune response? Because one interpretation of what you're saying is a person with cancer should never be calorically restricted. I don't know, that's going too far. I would say that I don't think that you can equate coloric restriction with the loss of autophagy or regulation of autophagy because I think they're not equal things because I think that coloric restriction is limiting tumor nutrients.

And so I think what that's doing in the context of cancer therapy needs to be better understood. I'm just not sure that we know what's happening there. If I'm hearing you, correct, but you're saying, look, it might be that we can't necessarily say that fasting isn't helpful in cancer because while it may be counterproductive from the standpoint of autophagy that may be offset by other things that are beneficial such as the reduction of overall nutrients and inflammation that accompany this. Exactly right. Yeah, to me, all of the areas of autophagy that have me scratching my head the most, it is this question of given that fasting is one of our most potent ways to stimulate it.

In fact, I would argue it's more potent than metformin, which is an AMP-AK activator, more potent, probably than exercise. I mean, it might be the most potent thing we can do to turn this amazing tool on. How do we think about using it in disease prevention and disease treatment, and they aren't necessarily the same thing. I completely agree. And in fact, I would ask a question of you. So there's a multiple efforts in the biotech industry to identify pharmacologic agents that are potent stimulators of autophagy. And I think their idea is, is that normal healthy people will take a pill, autophagy will

be turned on, and there'll be some fountain of youth type things. Yes, permitting is one of the things that people are talking a lot about, right? So why not cultivate the use of fasting instead? I will tell you exactly why, Eileen, and I love how you have fed into, it's almost like you can read my mind and know where I'm going to go with this discussion. I think a big part of it is we don't have the tools to measure the signatures of autophagy. In other words, if a patient comes to me and says,

Peter, I want to do whatever I can to enhance autophagy because I have now bought into the idea that it is going to basically protect me from every chronic disease. I would say, yeah, I agree with you. And they say, great fasting seems like a great way to do it. I'd say, you're absolutely right. And they say, well, how long do I need to fast, Peter? Guess what I get to say? I don't know. And I'll tell you in reality what I say. I say, well, look, I'm really sure that after about seven days of nothing but water, Atophagy is fully cranked. And I'm also really sure that if you just go 12 hours without a meal, you probably haven't done anything. Where I struggle is where between them. Now, I want to share with you some personal experience and I want you to weigh in on it. And then I think maybe we can pivot

off into actually kind of going back to what you and I spoke about over the phone five or six years ago, which is what would a molecular signature for a top which you look like? And again, I think this is, I put this in the top three most important translational questions in my field. In other words, as I think about the practice of medicine as it pertains to longevity, it's our inability to understand how to quantify the benefit of nutrient deprivation. In other words, our inability to dose it. That is our greatest. Certainly among our top three detriments to using this incredibly potent tool. So I fast a lot. I just finished a fast

yesterday, actually. So I used to do a thing where I fasted seven days every quarter. So four times a year, I would just do a water only fast. How hard is it to do that? It is not that hard. I'm going to be completely honest with you. I wish I could sit here and say, oh, I'm a real stud. Nobody can do it. No, no. Anybody can do it. I really think anybody can do it. It's not to say that there aren't moments throughout those fasts where it's sort of difficult, but you'd also be surprised at how resilient the body is. So yeah, not that hard. You have to make some adjustments. Obviously, you have to be very thoughtful about how much water you're drinking and how many electrolytes you supplement. There are a lot of changes that are happening in terms of electrolyte management and things like that.

But again, we certainly have the knowledge to know how to manage people through that. But I began to ask the question, right, which is, okay, is seven days a quarter the right dose? I'm convinced that it's a big bolus of autophagy, but is it frequent enough? What about three days every month? That's about the same number of total days fasted, but it's more frequent, but it's probably less potent. And the reason I sort of decided to try three days a month was I noticed that a couple of things happened

at the end of my fasts. So I always check my blood before and after one of these seven day fasts and there's a very predictable set of changes that occurs. Some of them that are really obvious. Glucose plummets. Insulin becomes unmeasurable. Euric acid goes through the roof, along with beta-hydroxybutyrate, of course.

Two possible explanations for the Euric acid going through the roof. One is the breakdown of nucleic acid. And obviously, and when I talk through, I mean, doubling of Euric acid. So when I talk through, I mean doubling of uric acid, so much so that I started taking alopeur and all during a fast to make sure I didn't get gout. Of course, it could also be, and I've read something that says that uric acid and BHB compete

for the same transporter in the kidneys, so there might also be a bit of a competitive blockade, but nevertheless, we have at least one possible explanation there. Endocrine function changes dramatically. T3 goes down significantly and reverse T3 goes up significantly, such that the ratio of them changes by 4 to 6 fold, which means you basically shut off metabolism, not surprising, explains why you become incredibly cold and tolerant during a fast, and also gonadot tropines go down. So you'll have all these really predictable things. But what I noticed was, I see virtually all of them, though not quite to the same magnitude

after three days, but not after two days. So that just got me intuitively thinking in a hand-waving way that three days was sort of the minimum dose you needed to really move the needle on a bunch of these other metabolic things, meaning the spike in uric acid, the bottoming out of glucose. So there's another thing that sort of happens. It's, as you turn more and more free fatty acid into ketone and turn more glycerol into glucose, you reach an equilibrium where your glucose is pretty much going to stay at

about three to four millimolar, and that takes about two to three days. And again, when we think about it through the lens, we've already discussed. AMPK must be up through the roof, M-Tore must be through the floor, and protein deacetylation must be off. I think my gestalt is that that takes about three days and obviously it gets greater and greater than the more you go. But what would be amazing is if I could draw tuba blood, send it to you after a three day fast, a four day fast, a five day fast, a seven day fast, a ten day fast, and get some sort of quantification. What is it that we would see in, now of course,

it's complicated because you'd probably want to accompany each of those tubes of blood with a muscle biopsy so that you could look at LC3, LC2 and things like that. So I'll stop on my diatribe for a moment and now turn it over to you, which is, where would you even begin to look for that signature of etophagy? And let's just start broadly with any tissue.

You can have blood, you can have muscle, you can have liver, you can have adipose tissue. How would you now create a dose effect? Well, we do that what happens at a mouse, let's just take a minute to discuss that. So, Misa Shima-led made a mouse that had a transgenic, LC3 EGIP protein expressed, and that could use that mouse. Tell folks what LC3 is, just because we're going to talk about this quite a bit.

LC3 is the protein that is attached to the autofagosome membrane and links to the cargo that ends up in the autofagosome. So this is a pretty mechanical thing, right? This goes back to your observation in the electron microscope. Exactly right. So LC3 is one of the key proteins attached to the auto-fagosome membrane and you use it to see auto-fagosomes because normally when autophages off LC3 is diffuse but when when autophagosomes form, the LC3 protein is attached to them and you start to see spots where all the autophagosomes are present. And so the mesoshema lab used that to assess autophagy in a living mouse. They fasted the mouse and they found that they could see the formation of all of autophagosomes throughout the mouse. And could they do this in like PBMC out of blood or do they need to use tissue? They did it with tissue. What they learned was that yes otophagy is turned on during fasting in a mouse, which wasn't surprising, but it seemed

to not be uniform across every tissue. So that was interesting. But we don't have a way to do that in people. All we can do, because this is involved making a genetically engineered mouse. So the only thing we can do in people would be to look at a tissue section and stain it for LC3 and look to see if there were spots. In other words, you could not look at LC3 conversion in white blood cells. You could, but there's another problem in that you could take PBMCs and do a Western blood for LC3 and LC3 gets processed from LC3 1 to 2 and the 2 form is a woman that's attached to the otophagosal membrane. So you could do a western blood of PBMCs to measure the conversion of 1 to 2,

but then 2 ends up in the lysosome and gets degraded. So the typical measurement of what autophagic flux involves measuring the rate at which LC31 gets converted to LC32, and then the rate at which LC32 ends up being degraded in the lysosome. And in order to see that flux, you need to block the degradation of LC32 in the lysosome. And in order to see that flux, you need to block the degradation of LC-3-2 in the lysosome with batholomycin or hydroxychloroquine. And so, you would only be able, by looking at LC-3-1 and 2 in PVMCs in a person that was fasting, you would only be able to infer autophagic flux because you wouldn't actually be able to measure it. It's a clue.

It's a clue. It wouldn't be proof, but it would be one could presume. I would expect you would see more conversion of one to two and then two going into the light of some. I don't think that would get you the answer that you need. Now, what if we had 100 volunteers who are willing to fast and subject to blood draws and muscle biopsies?

So you could use the muscle biopsies to actually quantify the flux and establish, let's say you could do it at different time points. So you had 100 people fast for different periods of time, three, four, five, six, seven days, et cetera. You've got tissue and you've got blood. What else could you look for? So again, could BHB be a proxy?

Could glucose be a proxy? I remember you once mentioning another organic molecule you had identified. You knew it by how many carbons it had and you thought it was ringed, but you weren't sure like what it was yet Have you figured that out? Yes, that was something that accumulated when we inhibited orthophagy and that was a Glucloronic acid so that would be I think you're going down the right road so I think What we can do and we haven't really done this yet would be to

look at metabolites because metabolism is so drastically changed. So if we can't look at directly measure autophagic flux in humans very easily because we don't yet have the proper tools, we could use metabolites as surrogate markers for the consequences of autophagy. That's exactly why I call it a signature as opposed to a biomarker, because I think it's basically, how do you use machine learning to take many metabolites? There's a bunch of things we know are happening.

We just have to integrate them. We know that loose scene is going down. We know methionine will be almost unmeasurable. We know what's gonna happen to glucose, euric acid. And then there's probably a whole bunch of other small molecules and things in the proteome that we don't yet know that are probably discernible from PBMC directly or indirectly through other things in the plasma.

And it just seems like a problem that is so ripe for a machine learning environment where you don't need that many people because you know what the gold standard is. You just starve them. And then you have the check, which is the muscle biopsy, which can give you some sort of quantifiable gradation. I mean, do you get the sense that that something in IRB would approve? It's invasive, you know, it requires biopsies and fasting and things like that.

But would they demand that you, hey, first you have to do this in mice? That's what I would expect. And so one of the things that we have been talking about is we've done some of this for probably not enough, is to do a metabolic characterization of a wild-type mouse fasted versus an autophagy-deficient mouse fasted. I think that would potentially identify the metabolic changes that were

autophagy-dependent. And I think that would provide some clues as to what to look at in humans, because the problem with looking at metabolism is you get an enormous amount of data, and it's very, very helpful to know what to look for. We may have a list of things that are obvious to look for, but... Right, but the fine tuning is going to come in the non-obvious. It's not going to be a regression model based on five things we know. It's going to be much more complicated. And I prefer not the underland post science. Yeah, taken on biased view and go

and honestly, I get asked about this more than any other translational problem. So the good news is I think there are a lot of people in the philanthropic community that would be interested in this, even if this is not a question NIH is interested in. I doubt NIH is interested in this problem, although that strikes me as odd, given how potent a tool fasting is and yet how we don't know how to dose it. And I think it's worth pausing on that for a moment, because that is such a stark statement if I'm correct, I believe I am, which is, imagine we had the most amazing drug imaginable. Imagine we had a proteos inhibitor for HIV and we knew deep down this could cure

HIV. The problem is we didn't know how to dose it. How long would we tolerate that ignorance? Imagine we had a drug that we knew could kill cancer, but we just didn't know how much to give or how often to give it. We wouldn't tolerate that for a minute, and yet infasting we have arguably the most potent tool, and certainly if not the most potent, probably one of the three most potent tools in which we can affect human health, and we don't have a clue how to dose it, or what frequency with which to use it. And I find that ridiculous.

So I'm actually really confident that if there were a really great proposal put together that would go from the animal model to the human model, it would be fundable. It would be fully fundable through philanthropic efforts. And so if nothing else comes of this discussion, I would love to plant that seed with you and think about what would be the right consortium of people to do that work. Obviously, there are lots of skill sets

that we'd wanna have involved in there, but I really believe that could be funded quite easily. And I think that the implication of that is as potent as anything else. Because again, here I am doing my three day fast every month versus my seven day, every quarter, versus five day, every quarter. Like, we just don't know. And it really is troubling to me. It just drives me insane. Well, I think for the general community, I think it's an important question, I think it's an important question, even for practical reasons, because you may be able to control your life to the extent that you can do all this at your own convenience, but

a lot of people don't have that flexibility. And so if they can be told that fasting for X amount of time is all you need to do, then the beneficial effects of fasting could be, there would be more people that could take advantage of it. Absolutely. And if you look at the work of someone like Walter Longo, who his assertion is, you can get most of the benefit without actually having to be fully fasted, but to do something that is like a fast mimic, where you reduce your calories significantly for a period of five days. Again, maybe he's right, but we have no idea.

We have no idea what the efficacy of that approach is versus a total water-only fast for five days. And it would be great to know because if we could demonstrate that you're getting 80% of the benefit doing a fast mimicking diet versus a complete fast, well, that opens the door to many more people who would be willing to do fast mimicry versus an outright fast.

And again, I think about this constantly, which is, I'm almost willing to do anything. I just want to know what to do. So I think that now is the right time to ask that question. Let me just digress a little bit to talk about metabolism. So, we know a lot about metabolism, essentially the field of not only just cancer metabolism, but metabolism in general mapped out all the metabolic, most of the metabolic pathways. But what we lacked the ability to do until fairly recently was to have a thorough understanding of metabolism and a living mammal. And so, Jaffa Benowitz and I have invested a lot of effort in developing technology to use isotope tracers. This would be C13 labeled glucose and amino acids and so forth,

and to deliver them to living mice running around and doing normal mice things in a cage, and then look to see how they're used and how different tissues use them and how there's nutrients sharing between tissues. Because when you're fasting, there's many complicated things going on. It's not just like there's no food and you're inducing autophagy, and that's that. That's the only thing that's happening in a vacuum, right? So you have dedicated nutrient stores, you have glycogen in your liver that's mobilized,

and that's dumping glucose into the bloodstream. You have a bad adipose tissue that starts degrading, your triglycerides, and then you end up with glycerol and fatty acids in the bloodstream, which then are taken up by the liver and so forth. So you have all these, if you're really without nutrients for a long time, then your muscle protein start being degraded, which is probably undesirable. That's dumping amino acids into the circulation so that you can maintain your survival. We have to understand all of that. Because it really occurs in different phases. Again, if you just limit it to humans

for a moment where we have a pretty good understanding of this, what's happening in the first 24 hours versus the next 24 hours versus the next 24 hours is very different. And George K. Hill's famous fasting study that 40 day fast on the healthy subjects really divides it into these phases. What's interesting is by about seven days into a prolonged fast, you pretty much reach a steady state. You've got a pretty consistent flux of triglyceride and defre fatty acid out of the fat cell. You reach a steady state level of beta hydroxybutyrate acetoacetate and glucose, such that basically the sum total of them in millimolar concentrations is about preserved to where you would be non-fasting like.

And so it begs the question, if we pos it that once you reach that steady state of seven to ten days, you're clearly in a fully turned on autophagy state, what's the switch look like? When you're 24 hours or 48 hours or 72 hours into that, are you 80% of the way to the benefit or just 20%. That's a jugular question. Yes. I think it would be fascinating to understand that. And I think that if you look back over history, almost all cultures have fasting as part of their history. And I'm thinking that that is not by accident. I think they must have learned by trial and error that this was a healthy thing to do. And so I

think that autophagy I would expect is playing a major role in promoting health in response to fasting. But I really think, well, maybe I'm sticking my neck out, but I think using fasting as opposed to trying to find some pill you could take is something that's easy to do. And of course, it speaks to the irony of it, which is if you took probably 1% of the budget that is being dispensed to find pills that stimulate a topology, we would actually be able to answer this question clearly. Absolutely. And actually just have a dose of Johnson.

And look, that doesn't mean these things can't coexist. I'm all for it, and I wanted to ask you, of course, about some of the other pills like rapamycin and metformin and the role that they might have and how we might be able to measure that. But again, this just strikes me as the most obvious question in the space of how to prevent disease. And it's like, you have this beautiful, beautiful tool. And you don't know what the dose is and you don't know what the frequency is.

If someone knows that they're susceptible to a neurogeneral disease, they're susceptible to a neurogeneral disease. Has anyone looked at those people to see if they engaged in some sort of fasting regimen, whether that was helpful or not? Indirectly, I mean, it's possible that that's been done directly, and I just am not familiar with that. I think what we've seen indirectly is dietary restriction, as opposed to just pure

color restriction, where you improve the quality of macronutrients, specifically around improving glycemic control. You can take people that are in an early stage of cognitive impairment and delay it and or reverse it through that type of nutritional intervention. Now, of course, that doesn't necessarily say atophagy is playing a role because that's doing a lot of other things.

It's improving glucose and insulin signaling in the brain. It's doing a lot of other things. And we're really starting to see the impact of metabolism in the brain. So that's not entirely clear. Indirectly, I would say there are, I think, some pretty interesting compelling pilot data that suggests that rapamycin is neuroprotective. And again, rapamycin, a very potent inhibitor of emtore, would presumably on some level induce atophagy.

I think it's a very interesting question as to what is it about rapamycin that induces a longevity phenotype? Rapamycin to me is the most interesting molecule out there because it is, I think, the only molecule that has demonstrated a longevity benefit across all four models of eukaryotic cells. So that's a really big deal that can't be ignored. But how much of that benefit is through autophagy? I'd like to turn that question to you.

How much of it is through inhibition of senescent cells, reduction of inflammation. Again, so it's very indirect, and it speaks to again. Again, I don't mean to sound like a conspiracy theory guy because I'm not, but it is a little frustrating that we have these amazing tools, but because they're not particularly profitable, you don't really have somebody that's interested in answering them. And that's why I again come back to, hey, I think these are answerable questions. B, I don't think they are billion dollar questions. I think they are really questions

that are amenable to the philanthropic community. And I think from an ROI perspective, it's hard to think of examples of where you could put dollars to work in research that would have a greater impact on human life. Right. And I think that our biomedical community is mostly focused on putting out fires rather than disease prevention, although I've seen a change.

I mean, I see at the NCI, the National Cancer Institute, a bigger interest in cancer prevention. So I think people are coming around to realizing that making people healthy or longer is probably more important than once they discovered to have stage four pancreatic cancer, what can we throw at it to make them live another two months. Yeah, again, it is sort of amazing to me how lopsided our resource allocation is with respect to that problem because you're absolutely right. We have spent probably a quarter of a trillion dollars in the last 40 years on the second question, which is once you have metastatic cancer, how do you live longer?

And we've done an analysis on this. So for the quarter of a trillion dollars that has been spent on that problem, on average for solid organ tumors, we have extended median survival by less than about a year since 1970, so almost 50 years. That's pretty sad when you think about the fact that there's not much evidence we've reduced the arrival of cancer. In fact, all we've done is basically come up with a second leading cause of cancer in terms of modifiable behavior, which is after smoking, it becomes down to diabetes, insulin resistance, and all of the metabolic dysregulations. So yeah, I'll get off my soapbox now, but again,

this is in many ways just a sort of a plea for help Which is I think there's interesting amazing opportunity to understand this So there's another component to this that worries me greatly so What we've seen you know, so a lot of this you're talking about controlling metabolism to preserve health through about controlling metabolism to preserve health through implementation of fasting and understanding fasting. But look what's happened to the American diet. I mean, there are people now that don't even recognize vegetables in the supermarket.

There are people that only eat prepared food. And so what we've seen is on the one hand you're talking about preserving health and all that, but on the other hand, the overall health of Americans is deteriorating. Obesity is greatly increased and has no sign of abating. The diet of Americans loaded with high fruit, doschorn syrup and diets that are disproportionate with prepared food. So the only way to globally improve the health of Americans or anyone else for that matter is to deal with both of these problems at the same time. And to me, that's probably the greatest line of reasoning that says fasting is probably protective against all chronic disease. Because if you look at the three main chronic diseases that account for, to our last analysis, 82% of deaths above the age of 50 in the United States, excluding COPD.

So if you take out the obvious smoking related death of COPD, 82% of death is attributable to cardiovascular disease, cancer, Alzheimer's disease, and complications of diabetes. That's pretty stark. There's no question that when you improve metabolic health, which you can do through fasting, you reduce the risk of all of those significantly. Of course, the question becomes how much of a role does a topology play in that? Specifically, you look at the example you gave of neurodegeneration that makes a very compelling case for it, probably also in cancer. What do we know about cardiovascular disease, by the way? Nothing comes to mind. I don't recall seeing anything in that area.

Yeah. I mean, my take on the literature is that the benefits of fasting in cardiovascular disease are primarily mediated through the metabolic health benefits of it. Lower glucose, lower insulin, lower home of cysteine in time, lower inflammation, primarily, as opposed to something that directly pertains to APOB or the inflammatory response to that. But again, maybe it's just there and I haven't seen it. What else do you think is going on? What travels with autophagy? Can we talk about synestense for a moment? Do we understand a sense of what's happening when an animal or a human is undergoing autophagy with respect to either sasp or just the overall senescent cells? Yeah, there seems to be complicated roles for autophagy

and senescence. There's evidence from the Naredalab that sasped the secretion of inflammatory factors that occurs during senescence is facilitated by autophagy. But then in the cancer setting, there's also examples where loss of autophagy limits senescence. So I think in the senescence area, it's still a little bit confusing and maybe a little bit context dependent as to what's happening. And then going back to the question about molecules, what do we know about metformin and autophagy? Do we know that in a fed state, if we give an animal or a human metformin,

we can still induce autophagy all things equal through just the AMPK activation. Yes, and I think that that's true, but I think that we don't understand over the long term what happens and what the consequence of having autophagy or not having autophagy is. So for example, we've never given metformin to our mice that don't have autophagy is. So for example, we've never given metformin to our

mice that don't have autophagy. That might be an interesting thing to do. What is the consequence of autophagy induction by metformin? It actually might be better to do that in a system where autophagy could be, wasn't completely gone, but could be toggled up. Do you have the ability to turn autophagy into an analog versus a digital, meaning where you can actually use gradations versus just on or off?

Yes, I think the mouse model where autophagy, expression of essential autophagy gene is controlled by, an SHRNA might be the way to do that experiment. And then what about rapamycin? What do we know about the use of rapamycin, which has been studied so liberally across, again, everything from yeast, flies, worms, mammals, uniformly extends life, potent inhibitor of mTOR, which would signal autophagy all things equal. Where do we see that relationship? So there's been a lot of discussion about using rapamycin or rapologs as autophagy stimulators,

but it's like what you said before, it does many other things and it's also immune-suppressive. Although that also depends on the dose, right? The Everalimus data suggested that it was actually immune-enhancing when given intermittently. Also have to remember that if you have a small molecule like rapamycin and you want to use it to preserve someone's health, you have to make sure that it's safe.

And I think that's when everyone backs away because if you've got cancer and they want to try an experimental drug and you've got no other hope and you're going to be dead in a short period of time, there's a bit of latitude in what can be done clinically to test whether or not there's a small molecule or some sort of drug that will be safe and possibly have efficacy. But when you're talking about prevention, it's a big problem. The drug companies are not interested in it because the amount of time it would take in the risk of...

Right, just a much narrower margin. By the way, that's exactly the reason we're in situation we're in. So, just to reiterate, we have a situation where I am not convinced that longevity as a game is going to be one on the back of extending the time you have a disease. I have never seen a shred of evidence to suggest that that is the answer. Everything in humans and animals points to the opposite end of the spectrum. Longivity is about delaying the time it takes

until disease comes. The implication of that is prevention is the single most important tool in the longevity toolkit. How do we reconcile that with what you just said? All of our pharmacologic efforts, the trillion dollars we spend on drug development, is all on the wrong side of the equation. It's on the, how do you live longer once you have a disease?

And I understand why that's the case for all the reasons you just said. And if that doesn't make the most compelling case for taking the best and safest drug of them all, which is fasting, and understanding how to dose it, and what frequency to dose it, I don't know what makes a better case. Along with exercise, by the way, I put exercise in that same category,

which is it bothers me that we don't really know how to dose exercise either. I mean, it's less of a problem, I think, because for most people, the issue is do more, but it would be really nice to know what the dose response is on different types of exercise, especially for people who want to do the minimum effective amount. So both from an exercise perspective and a nutrient deprivation perspective, there's no more low hanging fruit in terms of minimizing human suffering than understanding how these things work.

You should start a biotech company to do this. David Sabahdhine and I have talked about this at length, and he has constantly told me to do this. And the way we've thought about it, it's not going to happen because I won't do it. So someone else will have to do it. The way David describes it very eloquently is, right now, metabolic response to nutrients is a black box. And what David thinks, and I think he's right, is there's a multi-billion dollar opportunity in decoding the black box.

In other words, when we understand exactly what the response is to each different type of nutrient in every different dose and frequency, and we can decode that in the way that we can do with so many other biochemical processes, you can do everything. Because then you could actually develop drugs, probably they could actually do something. So there's a drug development platform that comes out of that, and then for my standpoint what I'm really interested in is simply just on the front lines as a sort of knuckle dragging doctor. How do you even just put this into clinical practice? But again, it's a high risk.

It's a lot of effort to do part of that. I think on the drug development side, there's a lot there. I don't think it's very high risk on the question I posed to you earlier. I think the, let's do the mouse study and identify the 50 metabolites, proteomic signature things that are generally going up with the topogy. identify the 50 metabolites, proteomic signature things that are generally going up with the topogy, and then let's shotgun that in an unbiased way against human subjects.

Like, I feel like that's a project in the tens of millions of dollars, not even the hundreds of millions of dollars. And again, if you add one year of life to each human as a result of that, that's kind of staggering. Right, and I think if you couple understanding mechanisms of metabolic delay of damaging diseases with surveillance for risk factors, I think those two things really need to be coupled together because you can even if you

define what the optimal diet and exercise and fasting regimen is for delaying the onset of disease, there are still unlucky people. And I think that we can't forget about them in the context of health and longevity and well-being, because you could have the healthiest diet and do everything right. Vogelstein has written about this, right? I mean, there's clearly a component. There's a stochastic component to this. And yeah, so absolutely. And again, I don't think there's any reason to believe that we couldn't be addressing both of these. Yes. I mean, I think that's a good way to do that. I think that's the reason to do everything. One always has to bear in mind that there is this other risk factor that no matter what happens

with understanding metabolism and fasting and good health practices, that other thing is still going to be there. If you have a BRCA1 mutation, that's a compounding and separate issue. So, in 2016, the Nobel Prize was awarded for basically the genetic elucidation of etophagy. What are the salient features of that award? What was it about Asomei's work that led to that award? So what Osemi did was quite profound and very creative. He developed an assay, well, he asked what are the, I mean, yeast requires nitrogen for

survival. And he asked what are the genes that are required for nitrogen survival? And he identified the, the, the central, the top G genes. And I always wondered whether if he was in the United States, would work like that be funded, because it just seems like it was an important question. But it didn't have such a clear application down the line. It was a bit too high risk to fund. Right. And you know, also the clear disease connection wasn't there and so forth and so on. And nonetheless, he did that. And I guess the point is that sometimes scientific discoveries are so basic

that you can't ever anticipate what it would ultimately lead to. And in this case, it led to something very extraordinary, but he probably had no idea at the time. And once they discovered these essential etymgy genes in yeast, then it was apparent that there were homologues and mammals and so forth and so on. How conserved are those genes between yeast and mammals? If you do a blast search, you can see them. I mean, not all of them, but the amino acid homology was compelling.

Wow. So, when you sort of think about the future of this, I think so much of what we've talked about kind of feeds into what your optimism is, but how do you want to spend the next 10 years of your career? What are the questions you want to probe? I think I would like to translate what we've learned about the role of autophagy and cancer. And that involves developing small molecules inhibitors to inhibit autophagy for cancer therapy.

And I'll at Kiliman and I started a company to do that. And what we're focusing on now is defining at the molecular level what the functional requirements for autophagy are in individual cancers. And this involves understanding the metabolic role of otophagy. Well, I won't cancer needs otophagy more than another. And then the newest connection is the connection to inflammation. When you inactivate otophagy, you stimulate inflammation. And this is what we've talked about earlier in the context of cancer. That could be a really good thing because the game changer in cancer therapy now is immune checkpoint blockade. In fact, what I was just talking about at the ASER meeting

yesterday was the particular patient that came to our cancer center and went through surgery, radiation, chemotherapy, and it all failed and her body was riddled with tumors and she went on a clinical trial for immune checkpoint placade and her all the tumors melted away and that was five years ago and she's perfectly fine. So what we have to do is make that work for everybody. And if inhibiting autophagy activates the immune response and can facilitate not people who wouldn't respond to immune check in my blockade to respond, then that would be critically important to do. I mean, let me think about that for a second. So when we think about the patients that are responsive,

and we really have two big targets, right? C-T-L-A-4 and PD-1. Melanoma, obviously, is a huge success story here because it is so mutagenic. It's interesting, I have a friend who has Lynch syndrome. So that's a familial syndrome where people are predisposed to cancer. He developed colon cancer when he was quite young, where people are predisposed to cancer. He developed

colon cancer when he was quite young, went on to develop pancreatic cancer, adenocarcinum of the pancreas, which is uniformly fatal. Almost without exception, he presented with an advanced state. So he was not even a surgical candidate. So the tumor had completely engulfed his mesenteric artery and vein, which meant he couldn't even undergo the surgical procedure, though it wouldn't have done much anyway. I had just read a paper six months earlier in the New England Journal of Medicine about, I forgot what the paper was exactly about, but it made me think that because he had Lynch syndrome and he has so many mutations, he might be a candidate for a checkpoint inhibitor.

So we went back to his oncologist and said, hey, can we get him on Ketruda? They said, which is an antipd1? They said, no, there's no standard for that. But we found a clinical trial. Actually got him in. He got Ketruda. That was five years ago. He's disease-free.

So you go from unresectable pancreatic adenocarcinoma to no pancreatic cancer, pretty remarkable. Now the question is, when I think about how broadly extendable that's going to be, it really comes down to how many shots on gold you get, how many mutations do you get such that you can activate these checkpoint inhibitors? Tell me how autophagy fits into that, because I think I'm missing the link of why enhanced immune non-specific immune response would factor into that. I know there's a link, but I need you to explain it to me.

That's exactly where we're going with research. I should have been at the ARC yesterday. So we know that tumors with a very high mutation burden respond better to immune checkpoint blockade. But it's not that simple, because there are patients with tumors that do have a high mutation burden that don't respond, and we don't know why. It could be, they've up-regulated some other checkpoint

that we can't yet inhibit or it could be some other reason they don't express class one or that the immune system can't see the tumor for some various reasons. And then we also know there are tumors that have a low mutation burden that do respond. They do respond, yeah. And so a big part, in fact we just got an NIH grant to study this is to make mouse models of cancer with low, medium, and high mutation burden with which to study. And then you can bang out a topogy. Exactly.

We haven't done the topogy part yet. We're just doing the generating the models using proofreading mutations and polymerase epsilon and delta to generate mice with cancer with various levels of mutation burden in their tumors. And this is so cool because we'll be able to ask basic questions like how many mutations do you need? When you have a low mutation burden, what can you do to make the immune system see that tumor? Do you need only mutations in a nuclear genome?

What about mutations in the mitochondrial genome? So one of the mouse models we made was to generate a mutator phenotype in the mitochondrial genome. There are human cancers that have a high level of mitochondrial genome mutations, whether that has any effect on anything is completely unknown. There's no reason why they couldn't be presented as tumor antigens. And you'd think of anything they would be more immunogenic.

I mean, they should be all things equal just because of their bacterial origin. That should elicit a much greater immune response. Exactly right. This is what this grant is designed to do. To generate mutator phenotypes in mouse models of cancer, so we can have a spectrum from low to really high mutations in the nuclear genome, as well as the mitochondrial genome, then to figure out the mechanism of response

to immune checkpoint placade to make cold tumors hot. And that's essentially what the loss of autophagy is doing by promoting inflammation. It's taking a tumor that is not killed by T cells that does not respond to immune checkpoint blockade and rendering that tumor responsive. And where do you put this in the hierarchy of optimism for the future of cancer therapy? I mean, to me, the interesting stuff in cancer therapy is getting more and more targeted and stacking more and more therapies on top of

each other. So this is an elegant example of stacking something that is clearly going to become a pillar of oncology, which is immune-based therapy, with something that frankly is partially metabolic and frankly partially more complicated than just metabolic therapy. So you've got, you almost add this to the layer of pieces of Swiss cheese. You start to stack on top. If you have enough of them, you're not going to be able to drop a pencil through the cancer, doesn't survive. That's right. So one of the limitations that we have with a immune checkpoint blockade is some of which I've already mentioned. We can identify who's going to respond and who's not going to respond.

And we have to extend the responder pool. But we have to be able to model that because it's very clear that a single agent says going to be a immune checkpoint blockade therapy is not going to help most of the patients. So how do we go about optimizing this treatment and having models where you can combine immune checkpoint blockade with other therapies to evaluate what is the optimal response is critical and that's what we're doing. And whether fasting influences this is completely unknown.

What's the role of autophagy in the immune cell itself? So either adaptive or innate, I mean, maybe both. So we know that one of the things we did was to turn autophagy off in a mouse and ask how that affected basic immune responses. And the answer was, everything in the short term appeared to be completely functional. And if anything, the T cells were more anti-tumorgenic. But if you go into the long term, if you knock out the essential autophagy gene,

only in T cells, for for example and look nine months later I think those T cells are not going to be very functional. But it really depends on how you design the experiment. For cancer therapy we want to know what happens acutely. When you're inhibiting autophagy you're going to be looking at things in the short term, not in the long term. So there's still a lot we need to do in that area. But in the short term for cancer therapy, the immune system seems to function well if not better in the presence of autophagy and abyssin. better in the presence of autophagy and abyssin. This just doesn't stop getting confusing because again, you would think that given the benefits

of autophagy and preventing cancer, one of them, you would think that that would only enhance innate immunity because of the role innate 8-immunity plays in cancer screening. Which again, I think just speaks to, we are still really scratching the surface of all of the different tentacles that come out of these tools. Something like fasting seems very simple, and it's simple, of course, to do, but it has such a set of pleiotropic extensions and benefits that it's very unlikely that it's about all or none. There's nothing black and white here. It's really all these shades of gray. That it's not intuitive when you look at them, what the net effect is because it's a little bit of this, a little bit of that. More of this than that, it's the balance of this versus that.

Yes, I would agree, and I think context is important too. When you think of all the big chronic diseases just based on what you've talked with us today about, I think we have to be really excited about Alzheimer's disease based on the model you've shared. I mean, that strikes me as an amazing opportunity because one, we don't have a single tool. Once somebody has Alzheimer's disease, I'm sure you saw the most recent, I don't know if you follow that literature, but we just saw two enormous failures in the anti-Mamoloid beta drug trials.

So we're back to kind of square one, which is not a single drug that works for this condition. If any disease demands prevention, it has to be this one. It's hard to make the case that fasting isn't going to play a beneficial role there, isn't it? I believe you. I'd love to. We have to test it now. We have to be tested.

But I think that when you look at the dramatic failures and preventing or delaying Alzheimer's disease, you have to ask the question, what is the root cause of that? And if you look at the approaches, all the approaches are designed to mediate a symptom. And the research is not yet gotten to the root cause. And I think that that is the reason for these spectacular failures,

is they're trying to treat a symptom of the disease rather than the cause of the disease. And when you look at the genes that are involved in neurodegeneration in general, they fall into a broad array of different categories. And so my thinking is that they're all doing different things, but there's some common denominator that has yet to be identified. And I think that until the root cause of disease is identified, just finding means to ameliorate the symptoms is not gonna be productive.

I mean, you know, I can sit here and talk about this for hours and hours. I wanna be respectful of your time because I know you've stayed an extra day to have this discussion with me, which I really appreciate. Is there anything else you wanna talk about today? Either is it pertains to your work,

something you're excited about in the future, or anything else that pertains to atophagy? I mean, I think that understanding in greater detail how autophagy impacts metabolism, we've done some of that, but I think there's way more to do. We have the technical ability to examine metabolic flux and amounts in vivo in normal, in starvation conditions and in response to different diseases and we're only at the beginning of doing that. And I think that that's something that we will continue to do that's something that we will continue to do and hope that we can identify new targets for anti-cancer therapy or signatures of metabolic problems, and we will continue to do that.

But again, I wanna return to the immunotherapy, I think in the field of cancer building on that huge gain, we can't take our eyes off that ball. Those are both very interesting and of course the former, it sounds like you really agree with David Sabatini which is there's an enormous opportunity to decode metabolism in a way that we should have done 20 years ago. I think we lacked the technology. I mean I think, so maybe I could take a minute to explain what we do, and that by using C13 or N15 labeled tracers, you can put them into a mouse, infuse a mouse with these tracers.

And by looking at where they go by mass spectrometry over different times, over different time points, you can see metabolism. And that was something that was never possible before. What is it because we've been able to label these things forever? What was it the mass spec didn't have the resolution before or what is it that why is it we couldn't do this 20 years ago? I guess I think they could do it with radioactive material, and they could do it somewhat with the technology

they had way back when. But I think that the technology now is far more sophisticated by using heavy isotopes. You don't need radioactivity. Now there are more mouse models of disease. You could even do this in humans. So Ralph Baradena at UT Southwestern is infusing these isotope traces into humans with cancer and actually measuring the metabolism

of human tumors. Wow, that's interesting. I did an experiment once in myself with doubly labeled water, which of course is a very simple version of doing that sort of thing to examine energy expenditure. I had a field they do in that. Oh. Yeah, but I think that the fact next week I'm supposed to go to a Keystone tumor metabolism meeting where all the experts in this area will get together and talk about this in detail, but this is a growing field. It's very exciting and understanding metabolism in mammals at a level we've never seen before in various disease states is tremendous. And then lastly, is it safe to say

that should there be a strong enough public demand and a philanthropic demand to go after that question we talked about earlier about sort of decoding the dose effect of fasting? Is that the sort of thing you'd be interested in working on? Yes. I was fascinated by what happened when mice didn't have autophagy and they died when they were fasted. And I am very much interested in fasting as a way to preserve health. I'm also interested in, as I mentioned earlier, the dietary part, because I think all of this has

to go together. It's not just how many calories you're eat or how often you eat them. It's what they are. So yes, that's something that's very important to me. Well, I mean, thank you very much. This was super, super interesting. And I know that folks are going to this. I'll probably pose a few more questions than even we had time to go into. But that's great. Your work is fantastic. Then I appreciate your generosity. Oh, there's been a lot of fun. I'm glad I took the time to do this. Thanks. Thank you for listening to this week's episode of The Drive. If you're interested in diving deeper into any topics we discuss,

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