The Peter Attia Drive - #110 - Lew Cantley, Ph.D.: Cancer metabolism, cancer therapies, and the discovery of PI3K

Episode Date: May 11, 2020

In this episode, Lew Cantley, Professor of cancer biology and Director of the Sandra and Edward Meyer Cancer Center at Weill Cornell Medical College in NYC, walks us through his amazing discovery of phosphoinositide 3-kinase (PI3K) and the implications for the care of patients with cancer. He explains various combinations of therapies being tested and used, including the possibility of pairing prescriptive nutritional therapies to increase the efficacy of drugs like PI3K inhibitors. Lew also explains the metabolic nature of cancer through the lens of his research into the connection between sugar consumption, insulin resistance, and tumor growth. Additionally, Lew provides some details about his exciting new clinical trial that is just now enrolling patients with stage 4 breast cancer and endometrial cancer.   We discuss: Teaching science through the lens of discovery—A better approach to learning science [5:15]; The metabolic nature of cancer, mitochondria, and a more nuanced explanation of the Warburg Effect [8:30]; The observation that convinced Lew to stop eating sugar [20:15]; The connection between obesity, insulin resistance, and cancer [25:30]; Sugar consumption and tumor growth—What did Lew’s 2019 paper find? [32:00]; Natural sugar vs. HFCS, fruit vs. fruit juice, insulin response and cancer growth [43:00]; Increasing efficacy of PI3K inhibitors with ketogenic diets, SGLT2 inhibitors, and metformin [53:30]; Lew’s clinical trial enrolling stage 4 breast cancer and endometrial cancer patients [1:07:30]; Pairing diet with drug could be the future of cancer treatment [1:09:30]; PI3K inhibitors on the market, alpha vs. delta isoform, and the possibility of pairing them with a food prescription [1:16:15]; What questions will Lew be focused on in the next chapter of his career? [1:22:15]; Lew's early work that ultimately led to the discovery of PI3K [1:27:30]; Studying the mechanism by which mitochondria make ATP [1:30:45]; How understanding the mechanism by which insulin drove glucose uptake into a cell got Lew closer to finding PI3K [1:38:15]; How Lew knew PI3K was important in driving the growth of cancer cells [1:55:00]; Lew’s unlikely observation of phosphorylation at the 3' position of the inositol ring resulting in the formation of phosphatidylinositol-3-phosphate [1:59:00]; and More. Learn more: https://peterattiamd.com/ Show notes page for this episode: https://peterattiamd.com/lewcantley/ 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, I'll 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 Lou Cantley. Lou is the Myer Director and Professor of Cancer Biology at the Sandra and Edward Myer Cancer Center at Wild Cornel Medical College in New York City. Lou's research has made significant advances in cancer research, stemming primarily from his discovery of the signaling pathway for PI3 kinase or PI3K in the mid-80s. And his results here have really touched multiple fields, including cancer first and foremost, but also diabetes and autoimmune disease. Lue is a very decorated scientist to put it mildly.

It would take me too long to rattle off all of the awards that he's won, but I'll leave it to the four most prestigious, the Gerdner Award, the Breakthrough Prize in Science, the Wolf Prize, and most recently the Horowitz Prize. Arguably the only two prizes that are not on that list would be the last-car prize and the Nobel Prize. And I think that many people suspect that Blue will ultimately be awarded at least the Nobel Prize, if not both. This is a podcast that at times gets very technical,

but most of the technical stuff falls at the end of the podcast. It's actually what we started with, but much like we did in a previous podcast, we're going to move that to the back end of the podcast so that we can jump right into the more concrete stuff that I think people want to hear about with respect to science. will treat what was the first 45 minutes of this interview as a technical tour to force of the discovery of PI3K and will move that as basically an appendix. So if you're listening to this and you really want to understand science and you really want to understand how discovery is done, stuff that I really like to hear about, by all means you'll just listen to this as it's laid out.

And if you really just want to hear about the implications of lose work as it pertains to cancer, then you will probably just listen to the first hour and 45 minutes or so of this and not the remaining 45 minutes. Before we jump into this, there are just two things I want to say. The first is I want to note, lose disclosures, lose is the co-founder of two pharmaceutical companies, AGOS and Petra. And he is also the co-founder of a company that is just getting off the ground called FAITH. That's F-A-E-T-H. FAITH is a company that hopes to pair nutrition with pharmaceuticals, and that will all make sense during the podcast.

These are all really interesting companies, but I think it's important, obviously, to list those disclosures at the outset. Secondly, I want to be sure that everyone understands that we have a discussion here that is very detailed at times and very tactical at times with respect to cancer treatment. And certainly, every time I talk with Lou, I come away with a new way of thinking about the care of patients with cancer. Please do not mistake what we are talking about here as medical advice. It is impossible to provide medical advice to someone with whom you do not have a doctor

patient relationship. And so, unfortunately, I hate saying that, but it is an obligatory disclaimer that I must provide. And whatever we talk about here, the combinations of therapies with respect to drugs for diabetes, drugs for cancer, and of course nutritional therapies really needs to be had in discussion with your physician. Actually, I lied and I said two things. There's one other thing I want to talk about, which is there is a cancer trial that is about to take off, that is a really interesting and exciting prospect through one

of Lewis companies. The enrollment for that trial is just kicking off now and we will list to that. Should anybody listening to this podcast either have the type of cancer that's being studied or know somebody who does and it would be great to know that people are able to more readily get enrolled in that clinical trial. So without further delay, please enjoy my conversation with Luke. Luke, thank you so much for coming over. Oh, it's great to be here, Peter.

You live like 10 feet from me here, and yet this is the first time you've been here, I think, right? Yeah, it took me five minutes to walk here. It's great to see you always. You're definitely one of the most generous people with his insights and with the work you've done. And I consider you an absolute mentor in how I think about these problems and biology. So we've been trying to get on this podcast together for a year, but it's a testament.

I was gonna say to how busy I am, but it's no, it's to how busy you are. I'm busy, but you are even busier than me. I do keep pretty busy. Well, there are a lot of things I want to talk about today. I think to set my own expectations, we probably won't be able to get through them all, which speaks in some ways to the breadth of your work. We're going to talk so much about cancer and metabolism and things here, but there's what we know today about PI3K, which I think would be a nice thing to spend a moment on.

But I also love this idea, like I have this idea, Lou, which is we should be teaching kids science, not the way you were taught biology as a 16-year-old, which was wrote memorization. We should simply teach science through the lens of discovery. So in other words, every biology class, even at the high school level and even younger, should be less about memorizing what it is that you see, looking at this mic, you up and memorize all the organelles or something like that. It's more of, let's tell the stories of science. Because one, if our goal is to train scientists, well, they're going to go and become experts in whatever they're going to go and become experts in whatever they're going to become expert in. We don't have to worry that in high school they're not going to learn enough content.

But if we're trying to screen for people who are interested in science, what you have to be screening for is the process, is the discovery, is the game, is the thinking that went into the experiment. It's the blind luck. It's the bad luck. And that comes out in the story of everything. I've yet to hear a single world-class scientist, and I have the privilege of sitting down with so many of them, tell their story without some element of good luck and bad luck combined.

CERN DIPITY. Yes. CERN DIPITY in science is absolutely critical that when you get a result that's unexpected, you should be laser-focused on understanding at the biochemistry, at the chemical level, why you got the different result than you expected to get. And following these journeys is, to me, a better way to weave the narrative arc of science. I mean, I think, frankly, it's not that there's no value in knowing that DNA is a helical structure, and it has these base pairs, and it has this backbone, but that shouldn't be the focus. To me, actually, walking through the fumbling of how those guys got there, that's a very interesting story. And that probably will serve a non-scientist better when they're done with science class,

because they'll have an appreciation for the process. And now I'm getting on my soapbox about it, it would be better if policy makers understood that that's how science worked, as opposed to trying to remember what they learned in a biochemistry class 30 years earlier. And of course, for the people who are going to go into science, I still think they're going to go back and learn the basics anyway. So we don't have to, through wrote memory, get that stuff into them. But it's this process of thinking and collaborating and walking down the hall and seeing the result,

as you said, that doesn't quite make sense and saying, well, wait a minute, there's something in there. There's a story, I mean, science is basically just one big mystery. It's really about curiosity. That's what I'm hearing in your story is it's less about curiosity. That's what I'm hearing in your story is it's

less about your innate brilliance and more about your obsessiveness, your ability to make an observation and not let it go. Yeah, if I get a result that suddenly doesn't make sense, to me that's more exciting. It means there's something more complicated going on than the simplest explanation for what I'm seeing. If the simple oxalination and fine, you publish it in some second rate journal, but if you've broken up a whole new field, because your simple explanation doesn't work and you figured out why it didn't work and what was wrong, then that's where most breakthroughs come from. When did cancer become such a high focus? So we met,

we met in 2011, two years after you, Matthew Vanderhiden and Craig Thompson wrote, what I thought was sort of the one of the most interesting papers I'd read on cancer in the journal science. It's hard to believe we're 10 years away from that paper. What was the lead up to your collaboration in that paper? And less about the paper, but more about thinking about cancer metabolism, because today, when I think of Luke Cantley, I think of cancer metabolism, when did that transition occur?

It was a postdoc joined my laboratory, Matt Vandroidon, who had trained with Craig Thompson, Matt's, an MD-PhD now in the faculty at MIT. But he had been obsessed about glucose metabolism as a graduate student with Craig. And in fact, I'd gotten one of his papers to review, which I was very impressed by. And so he suddenly showed up at my office one day saying that he wanted, he was interested in doing his postdoctoral work as he finished his clinical training. And at that time, a graduate student, Heather Kristoff, had been working in my laboratory and she'd made the observation that if she used tyrosine phosphorylated peptides to pull down a degenerate sequence context, it wasn't a single peptide, it was a whole mixture of billions of peptides

all at the same length. Biotinolated so you could pull down anything they bound to, and then use mass spec to identify all the things that came down. First of all, she rediscovered all the proteins that were known to bind to, that have SH2 domains. this is a domain that many proteins, including PI3 kinase, have that allow them to bind to tyrosine phosphorylated proteins. And so, in that one experiment, she rediscovered everything that had been published of all the things that bind to tyrosine phosphorylated proteins. But she's discovered the thing that bound better than anything else in the cell, or that was most massively pulled down,

was the enzyme in metabolism, pyruvate kinase. And that was shocking, and I remember her first response to that, was, well, the only novel thing I found here that wasn't already known, was this... The affinity for pyruvate kinase. Pyruvate kinase. And she said, well, that's a metabolic enzyme. And they're all boring.

And so I don't think I'm really going to work on it. And I said, no, no, that's whenever you get to result. You pull the loop. The totally unexpected result. That's the thing you should focus on. And so she did. And at the time that Matt was interviewing with me, I told him about her result. And he got very excited about that.

And the two of them worked together to show how that was working. So this is early 2000s, right? This was 2000 and mid 2000, like 2005, maybe. Matthew Vanderhaden was a classmate, I believe. I've obviously met Matt Sins, but I believe he was a medical school classmate or one of my close friends, Ted Schaefer, who actually I had on the podcast and Ted also was an MD PhD, did his PhD in Harold Barmas' lab, so it all kind of comes full circle. A lot of smart folks came out of the University of Chicago.

So anyway, so he maddened Heather into publishing two back-to-back papers in nature that both explained structurally in nature that both explained structurally how phosphatyracene peptides or proteins binding to pyruvate kinase could turn off its activity. I ended up also explaining why turning off the activity of pyruvate kinase was important to increase antabolic growth. And that is kind of the basis for why we decided to start agJOS. That Craig was interested in how metabolism was regulated in cancer cells. And we had made this observation that part of a Chinese regulation was important in oncogenic regulation and metabolic processes in cells. And so we proposed part of a Chinese activators rather than inhibitors as a way

to reduce tumor growth as one of the targets to go after at OJSS, we were starting the company. So that review, which I should say that Matt played a much bigger role in writing that review than Craig or I did. He was the first author. So yeah, that was pretty clear. When did the Warburg effect, when did you start paying attention to it? I mean, obviously it was around before you were born. So actually I mentioned F. Racker earlier. F. Racker was new

Warburg quite well and they overlapped in Germany. He was obsessed by understanding the Warburg effect, the Warburg effect being a result published by Warburg back in the 1921 or so almost 100 years ago. That if you chemically induce cancer in rats, the tumor that evolves takes up glucose and metabolizes it in an anabolic way much much faster than the tissue of origin prior to transformation. This was chemically induced cancer formations. And this also, this observation was that even when there was sufficient cellular oxygen to generate ATP much more efficiently through the mitochondria, it was as though the cancer cell elected to go this faster, less efficient route. That's right, the surprising ones all those, well there's plenty of oxygen, we've taken a tumor route,

we are in an oxygen environment, Why doesn't it make ATP through mitochondrial oxidative phosphorylation instead of through glycolysis? Why do you think the Warburg effect was largely forgotten in the 40s and 50s and 60s? I mean, it wasn't. It fell out of favor really not until late 60s or early 70s. And what was taking over at that time was evidence for virally induced cancers. V. Sarc, Harold Varmas in Mike Bishop. So once it was shown that viruses could turn on cancers, the idea that it was just an altered metabolism fell out of favor. Of course, the discovery of oncogenes of which V. Sarc was the first clear oncogen because

it told us that we've met the enemy and it is us, it is our own genes that are getting altered in cancer and so proto-oncugins were being picked up by retroviruses. Did anyone at the time think these two mechanisms aren't mutually exclusive? The expression of, or the amplification of, or the mechanism by which this viral injury to the genome can perpetrate and can perpetuate could be through this defective metabolism. I think people who thought about it deeply and who were trained as biochemists who were doing metabolism, which, if Rackerel was one of the prime examples, continue to realize you have to alter metabolism

to do cancer. You need to switch yourself not only from a quiescent state to a dividing state, but in order to divide and make a bigger cluster of cells, you have to drive glucose and amino acid uptake and metabolic processes converting those to proteins and lipids and DNA and RNA. So there was no doubt you had to turn up metabolism.

The question was at what level did metabolism play the role? Was it just a consequence of the viral transformation or the oncogenic transformation or did they work cooperatively in some more complicated way. So our P.B. R.I.P. R.I.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E.

and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E.

and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. and P.R.E. So that's why shift the de-emphasis back to there may be targets in metabolism that could give you an efficacy toxicity ratio for treating cancer. The truth is chemotherapy, as you know better than I, is really targeting metabolism. You're blocking DNA synthesis or other steps in metabolism as a way

of killing cancer cells. So that's, in a way, targeting metabolism is our first approach to curing cancer, and now we're back to doing it in a more serious way today. What was the point that you guys made that was so novel in that 2009 paper? Because it's a subtle point that I think you can easily miss. You could easily look at that paper and say,

oh, this is just another paper discussing the warberg effect. But you came up with a very clear explanation for why the Warburg effect might exist that was different from the explanation that I think I had looked at before, which was the mitochondria are defective. So I think the easy explanation for the Warburg effect is the mitochondria don't work. Therefore, the cancer cell has no choice but to undergo anaerobic glycolysis. You guys propose something different. Yeah, we proposed that you could even with totally functional mitochondria, you could divert

intermediates and glycolysis into anabolic processes by regulating steps in glycolysis. And so the biggest question, we know that if you have high ATP in the cell, it will shut off glycolysis. And that's a feedback that there's high citrate and high ATP, either one of those directly bind the phosphofructa kinase, shut off its activity. And the remaining glucose that comes in that gets phosphorylated, either goes to the pentose phosphate shunt, or it goes to glycogen storage, or it just goes back out of the cell again. So there's a break right there.

And so F-Racker was obsessed by how you relieve that break, because how do you get a 10 to 50-fold increase in glycolysis if you're making so much ATP? It should shut it off. It should shut it off. Shut it off again. So the idea that was defective mitochondria was a simple explanation. There's no other way to make ATP, so you have to make it through glycolysis. But the idea that there was something more complicated than that was really what we were discussing in that review that there was a way to regulate steps in glycolysis that would divert intermediates

in glycolysis from making ATP to carbon atoms of glycolysis and go into lipid synthesis, serine synthesis, glycine synthesis. So it was sort of like a mass balance argument. It was an energetics for construction argument. It was basically saying, look, the way to really think about this is cancers growing like crazy, it the way to really think about this is, cancer is growing like crazy. It's easy to talk about the energy requirement, but just think about the carbon requirement of growth. Right. So, one analogy I like to make is, imagine you have a river flowing through a valley, and you want to flood some fields in order to get enough water to grow plants. So, if

you put a dam here, then those other little canals that you've generated, the water level go up to flood into those other canals and allow you to grow plants. That's sort of what happens if you turn off pyruvate kinase, then you slow down ETP production. That means there's less ETP made, that means glycolysis at the part of glycolysis to continue. But instead of going to make more ATP, it goes off to make serine and glycine and ribose and lipids. And that gives you all the molecules you need to grow. Without shutting the system off, because you don't let it go all the way to ATP.

Right. And that allows the mitochondria to make whatever ATP you need. And glycolysis is now used to do antibiotic processes instead of making ATP. Right. Yeah. And it allows the mitochondria to make whatever ATP you need and glycolysis is now used to do antibiotic processes instead of make ATP. That still makes some ATP. But you can now rebalance that whole equation so that you can grow better rather than just make ATP. Now this had a profound impact on the way you thought about your life. You're one of the, I don't wanna say the few, but there are a lot of people who, a lot of great scientists, frankly, who, their professional work and their personal, the way they go about conducting themselves

in their own choices around food or whatever, there seems to be a disconnect there. Not the case with you. I remember the first time we had a meal in probably 2012. You were quite particular about what you ate and what you didn't eat and the thing that you were most careful about was fructose. So what is it about the work that you were doing in the 2000s, the early 2000s, into this decade we're in today, the beginning of the decade we're in today, that had you start to think about nutrition in that way. So I have to say that I made my decision about what to eat and not to eat before I knew

anything about metabolism. And it came from a simple empirical observation. So I grew up, I mentioned earlier, I grew up West Virginia. Backwards, the West Virginia. In retrospect, I realized that as I was growing up in the 50s, we were eating incredibly healthy meals. In those days, the most expensive thing to buy at the grocery store was sugar. And my grandparents, who I spent a lot of time working on their farm, raised everything they ate. They were substance farmers.

They had essentially no income. They sold some strawberries in the spring. My grandfather would plow fields for people to make additional money, but they had very minor income, but they lived off of everything they grew. The one thing they couldn't grow, for a while they grew some sugarbeats, but it was hard to get enough sugar. And so when my grandmother went shopping, essentially the only thing she would buy was flour and sugar, and one five pound bag of sugar had to last several years. And so, when she would make what she called a cake, we would call it a biscuit today.

It had essentially no sugar in it, because it had to be sprinkled in very small amounts. And so that, as a consequence, we were all very healthy, nobody in my family. In fact, I didn't know anyone who was overweight, was afraid to go to most most people lived like my grandparents. And that was in the 50s. What happened in the 60s was really what was tragic. It was all triggered by Castro, as you know, the story. And Castro's taking over Cuba and us boycotting sugar from Cuba and the anticipation that calls to sugar would go up even higher.

A German scientist that worked out a way to convert cornstarch into high fructose cornstarch, which could be made much much more cheaply than raising cane. It's funny. I was thought it was a Japanese scientist. I didn't realize it was a German scientist. I could be wrong on that. I would take your word over mine. We'll blame somebody from World War II. The irony of it is it's always We'll blame somebody from World War II. The irony of it is it's always someone that got hurt in World War II and the joke is this was their payback. Exactly.

And then Castro's paybacks, or Castro did this to us. We did it to ourselves as a truth, and we saw it doing it. And as long as I was where the primaries are held for primaries and where nobody's going to cut off taxpayer support for cornstarch or growing corn. But you had this profound belief. I mean it's I guess you got to experience something very few people do which is a natural experiment. Yeah so I watched West Virginia go from what I would argue in this 50s list, the skinniest state in America. I'd never saw anyone overweight. None of the kids in my school, none of my relatives,

none of them were overweight in the 50s. Most of them were very sad. And then along came high-fructural corn syrup, and suddenly the cheapest thing you could buy were sodas that normally at a six ounce Coke would be my total allowance for a week to buy one six ounce coke. And now suddenly you five, ten times as much for that cost. And so people would add then a very sugary drinks.

People would start consuming ten, twenty, fifty times as much sugar as they were consuming before. And over from the mid-s to mid 70s, I watched when I went back, I was at that time at Cornell, getting my graduate degree. And then I didn't go back to Western Union that often, but when I did go back, every time I noticed a dramatic change in that all my friends and relatives were suddenly becoming obese.

Now I just watched what they ate, and I realized they were consuming massive amounts of sugar. Many of them were consuming diet drinks, but then at the end of the day, because of their addiction, the sweet, like a gallon of ice cream per person. When I asked them, how can you eat a gallon of ice cream? They would tell me, if I don't eat a gallon of ice cream, I'll wake up two hours later and go to the refrigerator. So I can't sleep through the night without eating a massive amount of sweets. So at that point I realized this is a real addiction and I cut out drinking anything that was sweet and I cut out eating dessert.

So that was 1975. Didn't know anything about the metabolism of fructose or glucose. Just empirically, I saw the change in diet and the consequent massive change in obesity. So you basically have these two sort of separate stories, which is this is this observational journey of your life and then you have the work of your career that is bringing you closer and closer to cancer metabolism. work of your career that is bringing you closer and closer to cancer metabolism, when did you finally realize in the laboratory that this observation of yours had mechanisms to it that could go far beyond obesity, but also play a role in metabolic diseases, inclusive of cancer? What really brought it all together, and that's why I keep emphasizing insulin, as a postdoc

and ultimately starting my own lab, I kept coming back to how does insulin work. Whenever we managed to see this PI-kini's activity co-precipitating with Sark and insulin receptor, I realized that the same enzyme was mediating the effects of the oncogenes and also effective normal insulin signaling. And so that, by 1990, it was very clear in my mind that insulin was triggering cell growth in exactly the same way that oncogenes were. It were both activating PI3 kinase. And that suggested then a correlation between the two.

And particularly, as we began to notice, that the mutations, so many years later mutations in pythiricinies were identified, it was more than 15 years after we discovered the enzyme to mutations were picked up from Burt Vogelstein's laboratory. And as we began to explore what those mutations did, we realized they increased the ability of insulin to activate the enzyme. So this whole connection between everything that insulin does goes through PI3 kinase

and most oncogenes managed to activate PI3 kinase by either directly binding or indirectly activating it, not necessarily through insulin, but they were bypassing the need for insulin. We're going to come back to that very important observation in a moment, right? Yes. You know exactly where I'm going with the next level of questioning, okay? Right.

But it also raised possibility that being insulin resistant, which results in elevated serimentsal, because the pancreas has to generate more insulin. If your liver and muscle and fat cells are failing to respond to insulin, then more insulin has to be made in order to bring glucose back down. So insulin resistance is pretty much a silent disease, because you don't really know it if you go for an overnight fasting glucose measurement in the urine. Your glucose is okay, but if you do a glucose tolerance test with very few people do, you realize that the glucose

has goes up much higher, takes longer to come back down, and the insulin level is much much higher than normal during that process of adapting. So that's how in some resistance is defined, but I realize that high level of insulin, if we add it to our cells in culture, cancer cells in culture, it makes them grow better. And we've known that since the 1960s and 70s, we use fetal calf serum to make our cancer cells grow in culture. And if fetal calf serum doesn't work, we either order another batch, you know, another

lot from the company, or we just add insulin. And adding insulin always makes it work again. And so we've known for a long time that if you have high levels of insulin, you can make almost any cancer cell grow better than just pure fetal cancer, which already has some insulin, and it's variable from batch to batch. So in a way, it's been staring us in the face for years that insulin will drive the growth of cancer cells. And cancer cells tend to have more insulin receptor than the tissue from which they emerged. So in the process of tumors growing out, evolving, they turn up the expression of the insulin receptor. And that allows them to respond insulin

better. So as I kept seeing more and more data on this over the last 15 years or so, I became convinced that the active being insulin resistant or the state of being insulin resistant in humans sets them up to accelerate tumor growth. When I interviewed Sid, I don't know how long it's been probably six, eight, nine months ago, he shared something with the listeners that I think for many people is still hard to believe, which is smoking is the leading environmental cause of cancer. Obesity is second.

In fact, I think he even mentioned that in the documentary that was made after his book, you were in that documentary as well. Great documentary. It was not named after the book, was it in that documentary as well. Great documentary. It was not named after the book, was it? Was the documentary also called the Emperor of Almalades? Yes, it was. Okay, okay. Phenomenal. We'll link to it in the show notes because it's a real gem. I mean, it's worth every penny you've got to pay to download on Apple and watch that PBS special Ken Burns, of course, is a master. But Sid made the point there as well. And hearing you say what you're saying makes you think it's really less the obesity and

more the hyperinsulinemia that accompanies obesity in 80% of cases, which is high enough that you could easily, just from an epidemiologic standpoint, identify obesity per se as the trigger, meaning excess adiposity. But my belief, and I think yours, is that no, it's probably the hyperinsulinemia, which means if you're lean and hyperinsulinemic, you're worse off, and I think those data are becoming abundantly clear.

There was a paper published a few years ago out of Montessori, Albert Einstein, showing that, in fact, if you take breast cancer patients and separate them into overweight, slash obese, insulin sensitive because a lot of people can, if you put on peripheral fat, but don't have visceral fat, you don't necessarily become insulin resistant with overweight. So body mass index is not. Yeah, it's not enough. It's a first order term. That subset of overweight women who were insulin sensitive did not have an

increased risk of breast cancer, but the ones that were insulin sensitive did not have an increased risk of breast cancer, but the ones that were insulin resistant did. So that says a lot. And that paper also had data on lean women who got breast cancer and that correlated with insulin resistant lean women. And in many nationalities, particularly Asians, they may look very lean, but still have a lot of visceral fat. And so that I think we, our current mechanism of using body mass index is our correlation is not a good one. Yeah, it's just not good enough. And Mitch Lazar, in 2013, I believe, published a paper

showing basically the two by two of lean and not lean, metabolically healthy and not as a proxy for hyperinsulinemic. And it turned out that it wasn't the body mass that was the thing that tracked with health now The moment ago you gave this very eloquent explanation of the role of insulin and cancer Now four months ago in science you published a really interesting paper. What did that paper show? This is a paper in which was inspired by Mayor Bloomberg's attempt to ban the 40-ounce coke. So GA Young, a postdoc in my laboratory, who migrated with me from Harvard to Volkornel 2012-13, decided that she was inspired by Mayor Bloomberg and she would try to test the possibility that high sugar consumption would increase colorectal cancer.

She had worked with Bert Vogelstein as a graduate student, so she knew a lot about colorectal cancer and was working on mouse models of colorectal cancer as she migrated into my lab. And so we decided that we would design an experiment to test whether there was higher risk for colorectal cancer with high sugar consumption. And so it was really two questions there and my hypothesis going into this was the insulin level that if you go on a high sugar diet that you'll eventually come insulin resistant and that will drive high levels of insulin which may accelerate the growth of colorectal cancer. And in fact, we had data that said that what I just said was true. That in fact, yes, if we allow the mice to have sugar in their drinking water so they

could add lib feed as much sugar as they wanted on top of their normal child diet. How much sugar was in the chow? So the normal child diet, I don't remember exactly, but there might be 10% but you can buy these really high sugar standard American chows that are. Yeah, so we just gave them the normal child diet. And so because they were consuming massive amounts, they could drink as much of the sugary waters as they want. They drank three to four times as much water, sugary water, as if they just had water

without the sugar? In a consequence, they became massively obese, and they had accelerated polyp formation in the context of having knockout of the APC gene. So APC is the first gene in human cancer that gets lost to initiate the process of holorectal cancer development is but focusing on it. So the controls drinking normal water versus the sugar drinking waters both had the APC knockout. They both had the APC knockout. And what was the difference in polyp formation? So in that three or more fold sizes polyps, they were much more aggressive. And what about the

cancerous transformation? Well these mice, the polyps became so big that we had to sacrifice the mice because they literally could not digest food anymore. They didn't go into metastatic disease so we couldn't call it a progressive other than the fact that they were massive and it was beginning to occlude. I call it polyps. Now in this experiment you also tried to figure out what was playing the role in this, and you looked at glucose, so you could do this experiment with glucose water and fructose water, correct? That's right. But let me get to the next step first. Okay, okay. So that was nice and fine. It was consistent with my idea, but GA wanted to challenge me on the idea that maybe the sugar was directly feeding the growth rather than just making the mouse insulin resistant obese.

Oh, I'm sorry, because at this point, your hypothesis was all still through the insulin. This was all being mediated through insulin. Yeah, that was my hypothesis. So in order to try to always challenge people in my lab to prove me wrong, rather than prove me right, and because it's always learned more if my simplest idea is wrong.

Another great tenent of science. So she challenged me on this and Marcus Gancaldis is the other postdoc in my lab that worked with her and the two of them worked together. And they designed a diet that would not allow the mice to become obese. In other words, they were taking the total amount of sugar they were consuming. It was the equivalent of a 12 ounce cola sugary drink. And so on that diet, at least over the six months or so that they were on the diet, they really didn't gain weight. Maybe 5% increase in total calories consumed. So they did not gain weight. They did not become

insulin resistant. We didn't see elevation and see peptide, glycosolid hemoglobin. So by all characteristics, those mice were normal, they were not insulin resistant. And yet, they still had increased polyps by two to three-fold size. So at that point, that raised the possibility that the sugar might be directly feeding the growth of the polyp. And so we characterized that by using either radioactive glucose or radioactive fructose or carbon 13 label that non-radioactive but traceable form of carbon atoms in fructose or in glucose. And we also tried feeding them only glucose,

the same number of calories of glucose or the same number of calories of fructose, versus the mixture of glucose plus fructose. And they had to have both sugars, fructose and glucose before the polyps would grow faster. So that was really surprising. And do you think that that could be because the glucose provides the insulin and the fructose is, what do you think the fructose is doing in there? Because based on everything you said earlier, a glucose water alone, just a pure dextrose solution should have been sufficient, right? That's right. So that turned out not to be the case.

And the way to figure it out is again using carbon tracy, either by radioactivity or heavy atom. So we did both. And first of all, does it fructose or glucose get all the way to the colon? Question number one, it would seem highly improbable. But it turns out if you add, if you have glucose plus fructose, the glucose competes for the fructose for entry into the small intestine. And as a consequence, the glucose is not as efficiently taken up. And it makes it way all the way to the colon. If you give it in a sugary drink, I was just about to say, Lou, does this have something to do with it being in a liquid, so the transit speed is quicker? That's right. So if you gave the same amount in a solid food, it would never make it to the colon. But because it was in a watery mixture, it would transit the intestine fast enough that there was still fructose left.

Your experiment was elegant because you didn't have to do what my next experiment would have been, which is why I'm not a postdoc in your lab, is I would have done colonic lavage. I would have gone retrograde and seen if I could bathe the polyps and not even deal with the absorption, but I would have missed this detail. We wanted to replicate what? What's physiologic, of course. Yeah, the condition which humans do it. And other labs had already shown that, again,

giving out fructose glucose mixture as a liquid at a certain volume, the fructose would make it all the way to colon. So what we found if the fructose made a colon, the glucose got absorbed in the small intestine, but there was some local increase in glucose level in the bloodstream that's full that is. This is the first time I've ever seen this elegant demonstration of how you could miss in blood sugar a problem because by definition the faster it's going through

you the faster the glucose and fructose are getting through and bypassing the small intestine, the more likely you'll get actual colonic content of them, and the less likely you'll see glucose in the blood sugar, which is why the group that in the second experiment, who were not being given adlibitum access to it, didn't get obese and didn't develop hyperinsulinemia, but still developed the polyp phenotype. Yeah. As a side issue, along the lines you're saying, we actually found in a case where we had the water fed rather than a fixed amount to 12 ounce soda equivalent, that the subset

of mice that had the APC mutation actually had were protected from insulin resistance. It was the polyps reading so much as a sugar that it was protecting the mouse from insulin resistance. That is unbelievable. And at some point you'll blow through that and they'll become quite insulin resistant and get hypertrophic polyps. Yeah.

But in this case, we weren't allowing that to happen. It was a very small amount of sugar, equivalent of a single, drink a day, 12 ounce, sweetened drink. The real surprise was when we got to the molecular mechanism. And that was that you had to have fructose and glucose because the actual carbon atoms that were being used to grow the tumor were coming from the glucose and not from the fructose. And what the fructose was doing is when it went into the polyp, it was converted to fructose one phosphate by an enzyme called ketohexocainase, also called fructocainase. That enzyme is only found in

three tissues in any significant concentration, the liver, the kidney, and the gut. And so these polyps, like the normal gut cells, have that enzyme. That has been something of a mystery of why that enzyme exists in that location, but in any event it does, what happens is the fructose goes in and it gets phosphorylated by that enzyme. And it happens very rapidly. It's a very active enzyme. And that drops the ATP level in the cell because you're consuming ATP to phosphorylate

that fructose is coming in. Now I mentioned earlier that the thing with the warberg hypothesis is that in order to get glucose to flux at a high rate into a cell, you have to drop the ATP level. Well, this is a way to drop the ATP level. Instead of consuming it in some other way, you're consuming it by phosphorylating the fructose. There's an additional complication, or not complication, but intervention that is also important, which is once you start doing glycolysis at a higher rate,

process of doing glycolysis incorporates an inorganic phosphate to make the doubly phosphorylated, glycerol and win-6-pis phosphate. And so that additional consumption of inorganic phosphate drops a negative regulator of inocene diaminase, and that drops the ability to keep ATP synthesis going on in the cell. So the combination, too, drops the ATP level dramatically. And now the glucose that's coming in is flooding through glycolysis, but it's going into all these anabolic processes. It's being used to make lipids. We see all the label from glucose going into fatty acid synthesis

and serine synthesis and nucleotide synthesis going up fivertine volume. It's really quite dramatic what happens. But if you leave the fructose out, even though the glucose gets in the cell, it can't go through glycolysis at a high rate and so you don't get growth. You have to have both molecules. The carbon atoms are coming from the glucose. The fructose is basically driving the kinetics. Exactly. When you describe it this way, you sound crazy. I mean, you're the guy that nobody wants to talk to at the party. Your wife must be annoyed senseless by her crazy husband, who's got this hypothesis that now has so much emerging data behind it that says,

if you wanted to create a molecule to kill people, it's not just glucose. It's not even just fructose. It's put the two together. And guess what? Nature did that. Nature came up with a 50, 50 mixture of this thing. And if that weren't enough, she made it taste so good. Right. How do we reconcile this? Yes, so that's the part I like best. Because I think about everything in regard to evolution. Why do things evolve?

Why do we evolve to be addicted? Because you're ruining the party right now. Exactly. So I think this allows, if you think about humans 100,000 years ago, our metabolism hasn't changed to the last 100,000 years. So 100,000 years ago, our metabolism hasn't changed in the last 100,000 years. So 100,000 years ago, how often, during a typical year in a temperate climate, would you have available high amounts of fructose and glucose?

I mean, maybe in the fall. A month. A month or so. So you would get berries, ripen, apples, ripen. Keep in mind, these are very small berries, very small apples, this is before a domestication of plants. And so, as a consequence, what that means is at the end of the growing season is when most of the sugary producing fruits are being produced.

If you could eat enough of those and you could keep your appetite up enough to just keep consuming anything in place, anything available, you could eat enough of those and you could keep your appetite up enough to just keep consuming anything in place, anything available, you could put on weight. As you go into the cold season. As you go into the cold season. And if you put on enough weight, then you might actually survive to the next spring period when there's actually some roots to dig up to eat and keep going. And anyone who probably, almost anyone 50,000 years ago

who didn't put on 50 pounds or so in the fall would not be alive for the next spring. So a very strong requirement to gain weight in order to survive a period of time when no food is available. Yeah, Rick Johnson has written so eloquently about this and I'm blanking on the name of his collaborator who's an anthropologist and they even trace it back to which primates probably developed this first and they were primates that had left Africa, gone to northern Europe and most of them die off. It was only the primates that could develop this mutation and I believe it was a mutation in both Euro case and frictecainase. I could be wrong on that And I believe it was a mutation in both uricase and fructokinase. I could be wrong on that.

And I've interviewed Rick, and so we can go back and listen to that. But you had to develop this mutation, or else you wouldn't survive the winter. And then what happened was tens of thousands, if not longer, of years of strengthening that is what allowed those primates to then come back to Africa to basically become our descendants. So we as humans as a species have these mutations that would have served as well

when glucose and fructose combined were provided just at the right level. Yeah. So if you didn't anticipate that you needed to gain weight, your body would tell you to do it basically how it came about. And so it makes perfect sense. The extreme example is the hibernating bear. We call it the honey bear often, right? Because in the fall, not only do they eat every berry they can find in the woods, but they also climb up the trees and get to the honey bees, and they put on 150, maybe even 200 pounds within a period of two or three months and then they go into extremance on resistance and they

hibernate they fall asleep but because of being insulin resistant and having metabolic syndrome their ability to break down fats is impaired and that keeps them from quickly down fats is impaired and that keeps them from quickly burning up all the brantablism slows greatly they preserve the little bit of glucose they have for their brain and they feed their body off ketones and fats I assume. Very long period of time and then they survive so we don't hibernate but we probably became insulin resistant every fall hundred thousand years ago in order to survive these periods of starvation.

I mean, you've touched on this briefly from a public health perspective. I know you're not a policy maker, you're a scientist. Many would say it's impossible. Like we're just never going to see the day when sugary beverages go away, but the data are becoming harder and harder to ignore that there is something uniquely toxic, chronically toxic. People don't like the word toxic because they think it only has an acute implication.

Like ethanol can be acutely toxic, but I'm talking about the chronic toxicity. But the chronic toxicity associated with sugar, sweetened beverages, inclusive of juices, and things like that, right? It's not just a Coke. These data are becoming almost impossible to ignore. Yet they still proliferate. I believe their consumption is down. I don't believe people consume nearly the amount of sugar sweeten beverages that they consumed 20 years ago. I believe we peaked

at around the turn of the century, but we're nowhere near what you consume growing up in West Virginia. Is it possible to get back to that? Is there a solution or is it simply something that each person must be accountable for? Do you have a view on that, even? How you reverse policy, I think, changing cigarettes, smoking, there's a lesson to be learned there. What was the incentive?

How did the government make this happen, reducing advertisement, there's one way to do it. I mean, if you look at TV commercials, almost every commercial about food is about sugary food. And there are many of them tailored to young children to get them to buy the sugary cereals, to get them addicted to young children. You've got them for life, just like cigarette smokers.

You get them addicted at 15, 14. They're addicted for life and there's a market for you. So I think that's the peel we have to make. And the question is how do you do this one way taxing also helps? If sugar suddenly becomes like it was in the 50s, the most expensive thing that you bought at the grocery store, then maybe you would quit buying it. But of course most people don't buy sugar anymore. They buy processed foods that have sugar already added to them. By the way, looking at your experiments, was there any distinction between if you used sugar as the substrate versus hyphructose corn syrup? No, sugar sucrose, by sugar we typically mean sucrose from cane sugar, which can be

crystallized because it's a pure molecule. While hyphryctor's corn syrup is a mixture of fructose and glucose, that's 60, 40, rather than 50, 50. But in our experiments, the difference between those two doesn't make a whole lot. The fact that you need, if it's sucrose, you have to hydrolyze the bond is irrelevant to the kinetic acid described. That happens very quickly. So you don't buy the argument that some, I don't buy it either by the way, but that you

should eat your sugar in natural form, which is it should say cane sugar or beet sugar or something in the ingredient. And you can use this whole group of folks who believe that high fructose corn syrup is horrible, but quote unquote naturally occurring sugar is not. There's so much confusion around this, but I just want to know if experimentally you see any difference. No, I think whether sucrose, what shows the difference is whether the sugar is embedded in a fibrous fruit as opposed to being pure in a water. There's something about that liquid that is devastating. There are two things. One for colorectal

cancer, it is, as I said before, the fact that having it in a sugary water drink gets it all the way to the colon. If you had the exact same amount of sugar, but it was embedded in a fibrous fruit like an apple, then none of that fructose would make it to the colon. The transits to this intestine would be so slow that the sugar would have get absorbed. But does that increase the risk of cancer in other organs? No, I think this is absolutely unique to colorectal cancer. But I would argue, on the other hand, the difference between eating an apple and eating

apple juice, not only is whether the fructose makes it all the way to colon, but whether or not your glucose levels spike after consuming an apple, a whole apple eating it as opposed to apple juice. And the answer is with apple juice, you're going to get a glucose spike for sure. Eating an apple, you may get hardly any change in your glucose because it takes so long to break it down that the absorbance is slow. Well, and you might, you know, what I've observed, I don't drink apple juice, I don't drink beverages that have sugar in them. But what I notice is nothing has ever spiked my blood sugar more than a raisin on a

per mass basis. You took a fixed mass of raisins and you contrast it with a fixed mass of anything else. It's pretty stark because it doesn't have the water, doesn't have the fiber, it doesn't have anything else. And even if you ate, say, call it compare apple to apple juice, if you did equal grams of glucose, you might even get a similar area under the curve, but where it's going to look different is the apple juice,

which will have, you'll get a big spike, and the apple, you'll get a big spike, and the apple, you'll get a gradual, you're gonna have different insolents in response to those. So the glucose AUC could be the same, but the insulin response could be quite different. That's the point. And in my opinion, it's all about insulin.

Most endocrinologists worry about the glucose. I worry about the insulin. So really, there's three things you're worried about. You're worried about glucose, fructose, and insulin, but in how they coexist. Right. So colorectal cancer is unique. You can feed directly off the fructose

if you get it all the way to the colon through. But it has to be in liquid form. In liquid form. But the difference between liquid form, apple and apple juice is, as you say, the area in the curves is same, but the fact that the glucose isn't going up as high means you don't get as much insulin released.

Of course, if digested very slowly, the rate at which you're ambently burning glucose in your brain and muscle may be almost equal to the rate at which it gets absorbed in the bloodstream, in which case you hardly see any increase at all. That means no spike in insulin whatsoever. So that's the ideal situation is to never allow that insulin to get high. Now before we go into the Y, I want to go back to some experiments, some drugs, and something you said earlier. Let's go back to PI3 kinase.

Everything you said about PI3 kinase would make someone listening to this thing. If you could block it with a drug, could you block cancer? Well, obviously, you thought of that. So, tell us about drugs that block PI3K inhibitors when given to cancer patients. Okay, so by 1990, I was quite sure that everything insulin did went through PI3 kinase, and it was also quite clear to me that many cancerous emerged because of activation of PI3 kinase. Some pharmaceutical companies came to me and said, should we develop the PI3 kinase inhibitor? And I said, well, I can't imagine how you would be able to thread that needle of being

able to inhibit PI3 kinase without causing diabetes. Without causing severe diabetes. And unless insulin would apply enough to offset the inhibition of PI3 kinase, you would have to go off the drug because of a hyperglycemia. If you raise the insulin level and could override the PI2C and it was also going to activate the tumor to grow more. So it could be worse. Yeah, so I was skeptical and never in spite of all of our data saying the PI2C was driving a lot of cancers. I never pulled the trigger and starting a company or even

encouraging anyone else to start a company to make inhibitors of PI3 canes. The fact that mutations in PI3 canes were picked up in BERT Vogelstein's laboratory and colorectal cancer, and ultimately shown to be in many types of cancers, led the charge to actually develop an inhibitor. Because by then it was clear that other mutantirusing kinases, when they were mutated or hyperproduced in cells, could be drugged and those would be effective ways to block cancer growth. So with those observations, numerous companies, probably 15 companies went after projects

to develop in PI3 kinase inhibitors, I was still skeptical that you would be able to manage the insulin problem, but agreed, in fact, applied for and got funding from Stand Up to Cancer, American Associates for Cancer Research, to put a team together to try to figure out how to most effectively use those inhibitors as they went into the clinic. And I remember after we received that money, several pharmaceutical companies contacted me and said, well, you work with us.

We have a PI3 chemist inhibitor in human phasorontriols and they said, what's more, it doesn't call hyperglycemia, so it's going to be a safe drug. And I said, well, if it doesn't work, it doesn't call hyperglycemia, it's not hitting PI3 chemists, so no, I'm not interested in working with a drug that doesn't cause hyperglycemia. Ultimately, we decided to work primarily with Novartis that had a drug that we, several drugs, actually, that clearly called hyperglycemia. So obviously, convinced they were hitting PI-3 kinase and that that wasn't one target

effect. And the question then is, can you thread that needle? Can you manage the glucose level without getting insulin levels so high that they would reactivate piazzaric canyons in the tumor? And that had been the challenge. So we suspected, I should say, that in working with Novartis, we got them to agree that when the patient's got hyperglycemia, that the endocrinologist would judge who was not tolerable, that they would ensure that if the patient could not be

managed on the metformin, that they would have to go off the trial. Notice I didn't want them being given insulin. I didn't want them to be given insulin, or an insulin secretogog, which also raises serum insulin. And I should say that the endocrinologist who were called in and consult for these patients who were hypergoicemic on the trial, just agreed with me.

They said insulin is totally safe. They should be able to get insulin. And we should do it that way. But I was quite adamant. I said, well, I won't work with you on on these trials. Unless you exclude patients who have to be managed on insulin or insulin secretic ox. So Novartis kind of reluctantly agreed to do that. And all the way through their approval trial with that as their requirement, anyone who couldn't be managed who would need an insulin or insulin secretic ox

would be off the trial. And their drug got approved just in May this year. It took a long time because roughly half the patients who tried to enroll could not be managed on metformin. Could you also use SGLT2 inhibitors? So at that time, the SGL2 inhibitors had not yet been approved. So we're talking about 10 years ago when the phase 1B trials that were the lead in to the approval trial were being where we worked with Navaris on that.

But Navaris stuck with the initial requirement through their approval trial that everything had to be managed on metformer otherwise they had go off the trial and that's why it took them a long time to enroll enough patients to complete the trial. Now the SGL2 inhibitors are approved sodium glucose co-transporter inhibitors that reabsorb glucose from the ultrafilterate in the kidney. If you block that, then the glucose ends up in the urine. And so that's a good way to lower glucose

in there by lower insulin. So that tool wasn't available at that time. It's now available today. So we did a study over the last four years. It took a long time to do this, lots and lots of mice and lots of experiments to test whether in a mouse model for cancers, a variety of cancers, 12 different types of cancers. If you give a PI-3-Kennies inhibitor and use either a sodium glucose co-transporter inhibitor or put to patients on a ketogenic diet, which means that they only have about 8% of their

total diet as carbohydrate. And even that is slow release carbohydrate. The rest 80% is fat and 12% is protein. And we ask now comparing that formin versus insulin versus sodium glucose co-transporter inhibitor versus ketogenic diet. What works best in getting the PI3 kindness inhibitors did shrink the tumor. And I want to just pause for a moment. This is something you alluded to in passing 30 minutes ago, which is very important, which was insulin can go around the PI3K inhibitor when you block it.

And that's why just blocking PI3K alone wasn't going to be enough, is that correct? More accurately, we know that if you take a tumor that has PI3Kine's mutations, for example, X-Vivo. So we did a lot of this work in organoids from our human patients at Wal-Cornell, and we would take an organoid in a mutual tumor, for example, and give it a PI3-CUNY's inhibitor at the therapeutic dose,

and we could kill every cell in that organoid. But if we now added back the level of insulin that would be in the bloodstream, at the level of glucose that they would have experienced, and the level of insulin that would be in the blood stream, at the level of glucose that they would have experienced, and the level of insulin that we actually measure in the blood of the patient, 30 minutes after giving them the inhibitor. How high is the insulin level typically in that patient? So 10 nanograms per mil is roughly what we see within 15 to 30 minutes and by 90 minutes

it's up to twice that 10 to 20 to 30 nanograms per mill of insulin. And by two hours the insulin level is high enough that the glucose now in the bloodstream starts to drop. So that tells us that that massive amount of insulin is enough to reactivate PI3 kinase in liver and muscle. And so it's not going around PI3 kinase, it's just reactivating and spider the presence of the drug.

Because you can't develop a complete inhibitor for physiologic reasons. That's right. The dose that you can tolerate, yeah, fighting this battle. I see. I see. You completely turn off your insulin. So insulin still has to go through PI3 kin no matter what. You're putting up a porous dam because a complete dam would kill the patient. Exactly.

And insulin is tougher and then it's going to win. So let's suppose you were giving an estrogen receptor antagonist and you start getting toxic effects of losing estrogen. Would you decide that you would now give estrogen? Yeah. To correct that problem. And so that's what the endocrinology to say. We want to give them insulin to bring their glucose down. You're like, that's the whole thing we're trying to stop here.

It's what we're trying to stop is insulin because insulin is what's driving the activation. Those mutations in Piotrkenis will not activate Piotrkenis unless insulin is added to the tumor. And I recall, I think I even told this story. I may have told the story when I was talking with Sid on the podcast, but we were having dinner one night, and I talked about a friend of mine with breast cancer who was in a trial, and she was in an arm in one of the Boston hospitals, and she was on a PI3K inhibitor, and I think it was a phase two. She was the only woman who survived.

She's still alive today. The only woman with metastatic breast cancer that survived who on her own went on a ketogenic diet. I remember telling you this out of curiosity like, Lou, what do you think of this? Do you think this is just an odd coincidence? That was the time that you and Sid were starting these experiments. As Sid described it, that was sort of maybe the final clinical, the little pearl that sort of made you guys go off and do this. And that paper was published a year ago, right? That was

published last summer, last July. Last July, yep. So what was the efficacy of a ketogenic diet versus an SGLT2 inhibitor and metformin, were they equal? So if you did a run-in to the ketogenic diet, so they have an entire week on the diet, and then they're given the inhibitor, it's incredibly effective because by then you've depleted all the eye-cogen, all the relevant tissues. If you just start on the ketogenic diet a day before you give the first P.I. to ketogenic inhibitor, it's still pretty effective, but not as effective as the whole

week run into deplete glycogen. Is there any reason not to combine these? Ketogenic diet, metform, and S.G.L.T.2 inhibitor, P.I.3K inhibitor, as a F.U. to cancer? Four cancers, depending on this pathway, of course. Yes. Well, let me address the sodium glucose co-transporter inhibitor first, because that is almost as effective as the ketogenic diet in our mouse models. It keeps the glucose level not quite as low as the ketogenic diet, but pretty damn low.

And the importance, of course, is in both cases, it brings the insulin, the imid insulin, the serum drops dramatically. But the important thing is that every single 12 different types of cancers, some of them had PI3 connex mutations, others didn't, some were rast mutant tumors, didn't matter, some were, one was AML, and SIDS study. That's right, I'm retarget about that now. And I've skeptical that AML would have, because you never see a PI3 County's mutation at AML. But the bottom line was, there was a benefit in every single cancer we looked at. The reason we did 12 different types, we were trying to find one example of a cancer.

That would violate this principle. That would violate the principle. The bottom line is, I think, all cancers require some amount of PI3 counties to survive. And so, if we can keep the insulin level low, and insulin, of course, is the best way to activate the PI3 counties to survive. And so if we can keep the insulin level low, and insulin, of course, is the best way to activate the PI3 counties, if you can keep the insulin level low, and now turn off PI3 counties, you don't get that rebound of insulin, and the tumors just go away.

So ketogenic diet, if you can stay on it, would be my recommendation if you have cancer and you're going on a PI3K inhibitor. Sodium glucose-coach PI3K inhibitor. Sodium glucose coach transporter inhibitor, however, is easier for most people to take. It's a pill, and that's, I would say, the second best. And of course, again, it's hard because we're sitting here. We can't give medical advice to people.

I get asked this question all the time. It's a very difficult question to answer because I'm not managing the given patient, but cancer patients are among the most motivated patients you'll ever meet. I mean, you know this, you've been involved in these trials. They will do anything they believe has some modicum of science behind it that can increase their odds of survival.

Tragically, we're still kind of living in a world where the mainstream approach to this is a little bit convoluted. Obviously, one of the most significant concerns that many oncologists have, well-meaning, is that patients are gonna lose weight. I don't know how bad it is today, but certainly back when I was on the wards,

we would force feed cancer patients with insure. I don't know if you've ever had insure, Lou, but let me insure you, it is full of sugar. So now we're back to force feeding patients liquid sugar when they have cancer because we're afraid of them losing weight. Exactly. So one caveat or one concern which came out of the SIDS part of our trial is that at least in the mouse models of AML ketogenic diet alone. It was actually negative wasn't it?

Ketogenic diet alone and most of the tumors doesn't have too much. So just switching to a ketogenic diet, I think it's rare that that alone is going to work, maybe in some cases. But in the case of the AML, there was some evidence of some toxicity that came in the context, not normalized, the ketogenic diet, they're perfectly healthy. They live a normal lifespan, in fact probably live a little longer. But in the context of this AML model that Sid's lab was using, there could be some adverse effects, so we're cautioning people don't automatically go on a ketogenic diet if you have cancer unless you have somebody paying attention to potential side effects. Well, I had the luxury of sending, I mean, you've been so generous and I've sent many

tissue specimens to you from patients or friends, relatives of patients, and you've got your own Clia developed assay to look for insulin receptors and we've even made clinical decisions based on that, which is this is a patient who has lots of insulin receptor on their tumor. This is a favorable approach to cancer. This is a patient who doesn't, might not make that sense. How far is that type of an assay away from being a mainstream assay that any physician can use as part of their toolkit to start to customize cancer therapy?

To reveal my conflicts here, I do have a company I've started petroformaceuticals that is beginning a PI-3 kinase inhibitor trial. It will be combined with sodium glucose co-transporter inhibitor. I didn't even realize I'm bringing up all this stuff. I'm teeing it up for you here, right? Yeah, yeah. That trial will be starting any day now.

What type of cancer is Lou? Are you looking for an enrollment? So in phase one here, it's going to be a combination of a sodium glucose co-transporter inhibitor PI3-connys alpha inhibitor. Initially we'll be looking at solid tumors breast intrametral where you see high rates of mutations but we won't re- So breast intrametral in what stage of disease is this stage four? Yes, in stage four disease. So have you fully enrolled or? No, no, we're just, you know, Charles, just hasn't enrolled. So in other words, are there someone's listening to this that either has or knows someone with breast or

inner metrial cancer, who is stage four, how can they best find out information on this trial? So petropharmaceuticals, so of course this will be registered at clinicaltrials.gov, where you can find every single trough. So if you just go to clinicaltrials.gov and type in petrope.ert. we'll link to that for sure how many patients will you take in the phase one? I've forgotten the exact number but there depends on how many responses we see and so forth. But the dose escalation the dose escalation is going to be pretty quick because we already have we know what the dose is without a key. Each of these drugs you sort of know what

they do. We know we don't anticipate any So the hope is to get the phase two quickly. Yeah, we should get the phase two very quickly and the person, patients on the phase one could transition into the phase two. This is really exciting, Leo. So in that case, we will recommend that they have for breakfast because all these drugs, mostly these drugs require to eat breakfast before you take the pill because that limits some of the toxicity you should get from and increases the absorbance. But I think it makes a hell of a lot of difference what you

have for breakfast. So you'll recommend scrambled eggs and avocado and whole milk yogurt and not yogurt with sugar. Yep. Yeah. Bear a high fat yogurt and high fat yogurt with some crunched almonds in it or something like that. Yeah. Right. Now, Lou, is there any way to pair absolutely no juice, no insure. Yeah. In that time in the morning now, but evening, the drugable of already people low effective dose. So one might have some elasticity for what you had for your evening meal. Although I will tell you something, Lou, you probably see this thing in my arm, right? That's a glucose meter. Yeah. I know what a nighttime high glucose meal. I know what a dessert at dinner can do to

my glucose in the morning and my insulin and it's still high. So for people listening to this who want to enroll in this trial or even for you, Lou, I would just say the more stringent people can be based on what you're saying. If the hypothesis that's being tested is more insulin is worse, I think you're almost better off just biting the bullet and saying we're gonna do ketogenic diets. In fact, is there any probability in this trial of actually providing the meals to the subjects to make it easier, you know?

So I have another conflict here. I've also started a company that's making meals for dietary intervention in cancers in general. And this company also said, Mukherjee is a co-founder of this company as well. Called FAS Pharmaceuticals. How do you spell that? FAS F-A-E-T-H, which is a Welsh name for health, F-A-E-T-H. So this, like co-founders, are Karen Valston and Oliver Maddix from her laboratory, Ben Hopkins and Marcus Concolvus from my laboratory. These are a lot of the folks that were on that paper last summer.

Right. Ben was the first author, right? Sid Mukherjee also found her, this also Scott Lowe and Greg Hannon. Think I got every question. And the idea here is for patients who have cancers which the KD will be potentially a viable adjunct to what they're doing, the idea is,

let's pharmaceuticalize it basically. So is FAEF already selling product? No, it's just now getting started. We've gotten it funded and we're looking now for where we're going to be making the meals and how and whether we contract some of the meals out or we do them internally. This isn't going to be purely ketogenic, although we will focus on preparing the meals, make sure they have exactly the composition that we expect rather than just giving advice and hoping people find their right foods. But it's not purely ketogenic, in some cases, there's evidence coming out of Karen's

lab and also my lab that reducing serine levels can make some drugs more effective, producing methionine levels can make other drugs more effective in different mutational backgrounds. Same more about those two. I mean, those methionine and lucine might be two of the more potent activators of the target of rapamycin tour. Tell me about serine, that one's a bit. We don't hear about that as much.

So serine, some tumors we lie on synthesizing can either use serine in the serum or they can synthesize their own serine. So the enzyme phosphoglycerate di-dase, pH, EDH, is the first step in converting intermediates of glycolysis into serine and glycine synthesis. You need serine and glycine in order to make nucleotides as well as glutathione, so for redox, brinjial, and for combating ross rather. And also for building nucleotides, you need serine glycine. So you're trying to selectively deprive a cancer cell from nucleotide precursor?

That's right. How difficult is it to restrict methionine and serine in food? So this has been done, both in humans and in mice, diets have been tried to, and you can get this searing level down about 10-fold and still have quite viable human. And what about methionine? Similar. You can also have it. Without just restricting protein. Right.

We've been using diets for metabolic disease intervention for years, children, lack an enzyme in particular. Sure. Ketogenic diets for epilepsy and things like that. But even ketone urea. PKU. PKU. You need to reduce the final thinnellaline in the diet.

So this idea that you could get better clinical outcomes by changing amino acids, for example, in your diet has been proven clinically and in born-arris metabolism, so the idea that the same kinds of interventions, in fact, it is genetic diets, as you say, were developed for cancer, but for epileptic seizure syndromes. Yeah. Have you seen the data that have suggested,

this is a little changing gears, I don't wanna let you leave without at least some discussion of this, that in certain cancers when you restrict glucose, which would then imply potentially a ketogenic diet, you actually make the cancers more robust. And in particular in pancreatic cancer, have you seen some of these data? Actually, I've seen the opposite in pancreatic cancer. If you use a ketogenic diet in combination with the drug, so in our hands, putting a mouse that has a K-RAS, P53, mutant, pancreatic cancer, which is majority of human cancers have the mutational events. So in

that context, ketogenic diet doesn't really do anything, it doesn't accelerate, it doesn't slow it down. But if we combine that with a PI3, PI3, PI3, it never does anything to those tumors either. But we give the two together, the tumors completely disappear. So you have if we combine that with a PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, PI3, We're scratching the surface of that, right? And will these two companies, Faeath and what was the other one? Petra. Petra. Is the goal that these companies collaborate and start to pair diet with drug? Yes, we could certainly do that at this point, as I say, for Petra, we focused on the sodium glucose-coachransporter inhibitor because we think compliance will be easier to monitor.

And at this stage, we don't yet revved up to produce the diets that would allow those trials to continue. But yes, we could move on to the ketogenic diet and even possibly on top of the sodium glucose-cutter transporter inhibitor. But we'd have to do a phase one study. Yeah, you still have to go back to phase one. Phase one B. How many PI3K inhibitors are currently approved by the FDA? So there's the alpalisim, the vatastrag just approved in May, that it's specifically only the alpha isoform. Now that's the isoform that insulin activates and mediates everything insulin does. And that's the one that's caused all the problems with hyperglycemia. But there's also a delta isoform of PI3 kinase that's seldom mutated, but it's actually required for B cell growth.

And we discovered that back when we started knocking out PI3 kinase in a metapodic lineage cells and mice back in the late 1990s, a David Frueman, a postdoc in my laboratory, was doing those mouse knockouts and found that B cell lineage cells fell to thrive if you knocked out PI3 kinase. T cells on the other hand were fine. If you knocked out PI3 kinase, and ultimately it turned out to be the delta isoform that was critical for the growth of B cells. So when Delta inhibitors who were developed, they went into B cell lymphomas as the first possible place to work since that ends. I was already known to be critical in B cells.

And they turned out to be effective. Adel and Lysub was approved about five years ago. It's Delta specific pediatric antithubitor. Who made that? That was made by Gilead. Oh Gilead, okay. five years ago, it's Delta-specific pediatric antithinic inhibitor. Who made that? That was made by Gilead. Oh Gilead, okay, yeah. And then more recently, a drug called Kapanlisub was proved about three years ago by Bayer. It's a drug developed by Bayer. And which isiform is that? Again, It also hits Delta, but it also hits Alpha

So the alpha one is the only one that causes the hyperglycemia. Yeah, that's the one that my friend was on by the way Right, but that was approved in May and that seems to have the most ubiquitous application doesn't it? It's certainly where you see the most mutations and of course the breast cancer is Far more numerous than lymphoma. Yeah, but that's the one that's going to need the most management of the hypergoysemia and the hyperinsulinemia. Is that message reaching the oncologist? Slowly, I think they're beginning to understand what we're saying, why it's important to keep the insulin down. The problem, as you know better than I, that we were so specialized today in medicine, that people commit to go into either oncology or endocrinology, and from there on, their studies are completely, they're studying completely different things.

And if you have an endocrine problem as an oncologist, you don't try to solve it yourself. You call in an endocrine consult. So your brain isn't really even focusing on how to solve a problem because you have an expert who can do it for you. And the problem is the endocrinologist are not really that alert about what's going on with cancer. Their job is to make sure the glucose is in line. Yeah, sort of, I think it's time that we either, I mean there's really two ways around this. I see, one is you sort of create a new specialty, which is metabolic oncology.

Today, if someone wants to study oncology, they choose between surgical oncology, medical oncology, radiation oncology. Of course, the challenge is you have to differentiate very early. You don't do oncology first, then surgery, then medicine, then radiology. You do radiology, or medicine or surgery, and then subspecialize So therein lies a problem. How would you become a metabolic oncologist? So the other option is basically oncologists learning more about the metabolism of cancer so that when it comes to managing understood and predictable complications of these medications, which are actually desired features as you point out. If you're using

this drug correctly, you should see hyperinsulinemia and hyperglycemia, and you should treat it accordingly, which reminds me, by the way, how often would the alpha isoform, does that drug need to be administered, or in your trial? The decision of how frequently the dose is based on PKPD, the drug level goes into the bloodstream, and how long it stays high and following how quickly it declines. And so most of these drugs that make it in the clinic can stay at their therapeutic dose for three or four hours.

So a lot of the companies want to do by two drugs a day, one in the morning, one in the evening. And what's the total cycle? How long are you being given drug? How long are you giving a rest from the drug? And so, for example, the Nevada's drug is daily. It's once a day daily after breakfast on a full meal. And why does it need a full stomach? Because otherwise absorption problems, you don't get as good a PKPD if you eat your own empty stomach, and you get more side effects, diarrhea or nausea, whatever if you try to do it on an empty stomach.

But there's no recommendation in an approval trial as to what you're supposed to eat for breakfast before you take the drug or whether you take it within your or apple juice or water. So. Hence the importance of a company like Fayette, they could start to standardize that because look, everybody wants to do the right thing here. And wouldn't it be great if as a doctor you could write a prescription that's not just covering the drug, but covering the food? Yeah.

So that's what we're anticipating at some point. If you can prove in a clinical trial setting that you need this particular food for the drug to be affected. To maximize absorption, minimize symptom side effects, and most importantly maximize efficacy. You should be able to get that food paid for, the delivery of the exact food that matches what your need is. Think about it. What a trivial cost it is compared to the drug.

I mean, you could send people caviar spread all day long at one one hundredth the cost of a drug. Exactly. But you have to prove it. You've got to prove it in a phase three. Well, don't get me started on that. I mean, think of how many drugs have been approved that haven't proved Jack. I mean, it's ambience should have been approved.

Anyway, don't get me started on the drug approval process here, but I'm completely with you on that. Lou, I want to be kind of mindful your time. There's kind of one last theme I want to explore. So you're going to be 70 this year, right? Which is kind of hard to believe because any I already passed you already passed it. So you don't look a day over, I don't know, 56. You've got this ageless phenotype to you. So I don't want to for a moment suggest your career is anything other than continuing to go up in terms of the work you have to do. So as you think about the next chapter of your career,

what is the question that you are most obsessed with? What is the question you want to spend as much time as you can trying to answer that currently really don't have an answer to? That's hard for me because I'm the most unfocused. In some ways, I'm compulsive about getting the answer of every question that I get curious about. And as you can tell from this interview, I have an opinion on how everything works.

And having never taken any biology course, I have no prejudice about how it should work. I just, so for me, everything that I run into biology is, here's a mystery. I don't bother reading the textbooks because I know they will prejudice me to the wrong answer. And so I'd like to get to the bottom of what's really going on. But the bottom line is that I get absolutely brilliant post-docs and graduate students to come to my laboratory. I allow them to do anything they want to do.

And if it obviously gives them advice and help interpret the experiments, but this has kept me in so many different fields for 40 some years as I've run my own laboratory. It keeps my mind continually alert about every possible thing. So, yes, epileptic seizures. It turns out probably most of the people have epileptic seizures. If it's a picture-cAA mutations as a mosaic, as a mnurons and a brain, ketogenic diet was developed to minimize that.

It makes perfect sense. Keep insulin down. If a picture of a mnuron is so, you're going to get less firing of that neuron that's going to solve the problem. But that wasn't how the ketogenic diet was developed. So my curiosity, just studying everything that's which PI3 kinases involved at some level, could keep me busy for several years.

But that's not the only thing I'm interested in. I'm interested in whatever curious observation that comes up from experiments in my lab, it can't be easily explained by current knowledge. To me, that means there's an opportunity to break open some new explanation for some disease. So I'm willing to work in almost any disease that's not yet solved and there's an opportunity to figure it out. I'm a chemist. So to me, unless I understand some of the molecular level, I don't really understand it. But I think with PI3 kinase, I really understand it to molecular level. I think that's an understatement. Very true.

There's still some mysteries there. I should say that we're about to publish a couple of papers, hopefully over the next six to nine months, that are going to completely change everybody thinks about PI3 kind of regulation. So there's still some major things to learn about this inside. Well, there's so many of the things I'd love to speak with you about, Lou, but I know that some of them are still work that you're doing, and therefore I know you would prefer to wait until things are a little further along to talk about them, but we'll have to

come back in a year or so and do this again, because there's so much other stuff that I know we've talked about informally about cancer that is, I want to believe it's where the field is going to start to go. I want to believe that because I get so many emails from people who say, you know, my mother has cancer or my husband has cancer and they're being offered this therapy and it's a very standard therapy. The patient is progressing through it. What metabolic options are there? And people like you are, people like Sid, people like all your colleagues in Matt and Ben and all these guys,

I mean, you guys are the guys that are kind of driving that forward. And you've made a lot of progress in a decade. It doesn't feel like it sometimes. Like sometimes I feel like it's not going fast enough, but I think having a discussion like we did today makes me realize how much has happened in a decade. And it's actually quite a bit, I have no doubt that you're going to be here a decade from

now working just as hard. Well, my real motivation, of course, is to convert every observation we make in the laboratory to actually change in practice for whether it's diabetes or cancer or what other disease that we begin to understand in molecular mechanism, if we don't convert those observations into actual change in behavior or change in practice or change in drug metabolism and diet, then in the end we failed. And so that's my goal. And as being a cancer center director, people sometimes listen to me now when I suggest this is the way, this is the trial we should do,

and this is the way it should be designed to test the idea. For me, it's spectacular being here in New York City, a Wal-Cornel New York Presbyterian where we really can translate these breakthroughs into new therapies. That's what my goal will be over the next 10 years. I hope you enjoyed that discussion with Lou Cantley, and I hope you found it half as interesting as you can probably tell I did. As you may recall, at the outset of this podcast,

I said that the first 45 minutes, which was a much more detailed technical description of lose initial work in the 70s and 80s that led to the discovery of P.I. 3 kinase, that content would be bumped as almost an appendix to the end of the podcast. That's what you're about to hear now. I hope you enjoy what was originally the first part of my discussion, but ultimately the appendix to this fun discussion with Luke

Helix. There are a lot of things I want to talk about today, but I do want to start with the discovery that you led that probably most people would associate with you. If they go to your Wikipedia page, that's probably the first thing they're going to see, although I haven't been to your Wikipedia page in a while, so I don't recall. But let's talk about PI3 kinase. How do we define it, by the way, tell people what it would be animals? So PI3 kinase stands for phosphoenocytide-3 kinase. And the three means that phospho

relates the three position on the inocytol ring, which is the head group of this lipid. And so in the mid, early mid 1980s, phosphatotol inocetal phosphorylation, which was known to occur from 1949, the purpose of it was totally ambiguous. Why did this lipid get phosphorylated? And in early 1982, 83, there was a breakthrough with the discovery that the phosphorylated form

phosphatotideolanocytol was phosphated to four and the five positions of the anocytol ring. By the way, anocytol is hexahydroxy, cyclohexane. So there's six hydroxys. One of them connects to the glycerol backbone. Hesitol is hexahydroxys, cyclohexane, so there's six hydroxys. One of them connects to the glycerol backbone, and then the others are potentially available to be phosphorylated. Anyway, the four position and the four plus five position were both identified the year

I was born, 1949. And the purpose of that was not known. It was called the futile cycle, maybe a way of just getting rid of phosphates or something. We're getting rid of ATP. Yeah, yeah. It was phosphorylated and deep phosphorylated and sort of like an ATP sink. And then the idea, the observation that this phosphorylation

could be stimulated by various GPCR pathways and growth factor pathways got people thinking about what it might be doing and it tended to correlate with calcium elevation. And so the breakthrough paper showed that in fact when cells are stimulated with certain growth factors like EGF or a certain subset of GPCR activators. And those stand for G protein coupled receptors and EGF just for folks. Epidermormal growth factor.

Okay, so all growth. And so these signaling pathways would activate the hydrolysis of that lipid that the anositol 145 trisphosphate, it comes off when you hydrolyze it away from the lipid, turned out to regulate calcium release from cells. And that was a huge breakthrough, because no one knew how calcium got elevated and that explained it. So in the meantime, it was not working

on that particular pathway. And let's pause for a moment and remind everybody, you trained, you were interested in science from a young age, but you were sort of deciding, I think, a little bit, if I recall, between chemistry and biology, you were very interested in both, I think, a little bit if I recall, between chemistry and biology. You were very interested in both, correct? I was a pure chemist.

The last biology course I took was in 1964 when I was a sophomore in high school. So you very quickly declared chemistry your obsession? I hated biology because at that time, at least in the backwoods of West Virginia where I was growing up, biology was just a bunch of descriptive memorization. And so I was bored with that and decided I would never take another biology course and I stuck to that. I was pure chemistry, mainly organic chemistry, and then I switched into physical chemistry, but was totally uninterested in biology at all.

Until I took one semester of biochemistry and realized maybe there's something interesting going on in biology. So I went to Cornell and it's a good to get my PhD and there I focused on bio physical chemistry. It was a chance to apply chemistry to questions like how do you get molecules across membranes. And that's what I worked on. How do you synthesize ATP in the mitochondria or chloroplasts? So that kind of the slippery slope of going into biological questions that could be addressed by simple chemical questions or physics questions.

Now, what got you interested in the mitochondria so early? I read a paper when I was an undergraduate that was an idea that Peter Mitchell had proposed. That the way you make ATP in the mitochondria, and we're chloroplasts, was using a proton gradient. And his idea was that that gradient would allow you to pull the protons in one direction, the hydroxy ions in the other direction, and remove water from phosphate plus ADP and condense that into ATP. That was just a very simple physical property that if you could move those two ions in different directions it would produce ATP. In effect the idea

was not correct. It was correct that ATP was made from proton gradient, but the actual mechanism was turned out to be different. There are actually three Nobel Prizes given for how ATP is synthesized in the mitochondria. I wasn't aware that there were three separate Nobel Prizes. Do you recall who won all of them? So Mitchell obviously, yeah. Mitchell obviously, yeah. Mitchell, not one. It didn't. It was the second one by Boyer on the, figuring out the actual mechanism by which this occurs. And then a third one from Peter Walker

who figured out structural basis for how this all worked. Oh, I didn't know the Walker one, okay. He was a crystal structure and did crystal structures. And I met all of them when I was a young man before coming to Professor. And so it's, that was very invested in that, and that's what I worked on. For the person who's listening to this, whose's a world is not necessarily wrapped up in this,

which is most people, it's worth pausing for a moment on how much is involved for the Nobel Committee to recognize one body of work that says something that three times they would recognize a different angle of the same body of work, probably speaks to why there is so much to this day complete interest in fascination with the mitochondria, beyond just the obvious, the energetics, which is what was initially how we would think about it. Well, it was a fascinating question in the 1960s. This was the biggest mystery because we knew how you made ATP from glycolysis, but how you made it in the mitochondria and how could the mitochondria do it so much more efficiently

than glycolysis. That was the big question when I was a graduate student. I think this podcast may represent the point at which mitochondrial topics exceed all others. It also speaks to that nature. So Mitchell, the great. Tell me about how you interacted with Mitchell. How did you come across him? So he came to give a talk at Cornell. Okay. And I should say, F. Racker, F. Racker, who was chaired the biochemistry department at Cornell and who I got to know as first year graduate student because I was getting mitochondria

from his laboratory in order to purify the enzyme that's synthesized ATP. Every time I would be waiting for the centrifuges to stop running, he saw me, he would grab me and pull me into his office and start throwing out ideas of how he thought ATP was synthesized. And the truth was that at that time, he did not believe Mitchell's Chimiazmotic hypothesis, Peter Mitch Mitchell was proposing. And so as a consequence, I got all of these ideas that he was throwing at me.

I personally believed that Kimi-Azmotic hypothesis was correct. Between those discussions, as I continued to, I was working in physical chemistry, I was in the chemistry department, not the biochemistry department. But the fact that he would spend so much time with me, even though I wasn't even in his department, to me was quite flattering and really exciting that this world quality person had done it was willing to talk to me. So he went on because he was trying to prove Peter Mitchell wrong. He completely purified every component, generate a synthetic membrane, reconstituted

the proton pump into that membrane, generated a proton gradient, and was able to quantitatively synthesize ATP. And it was the year after he published that paper saying that the chemiosmotic theory is actually correct. But he did the definitive experiment that the prize went to Peter Mitchell. Today, I almost guarantee they would have that the prize went to Peter Mitchell. Today, I almost guarantee they would have shared the prize. Mitchell had the idea, Rack approved, and it was correct, even when he was trying to prove that it was incorrect. So that was an interesting story that Mitchell was invited to come and give a talk at Cornell just at that time. My last year as a graduate student. And so he met with me and my advisor, Gordon Hamis.

And I went through my entire PhD thesis with him and F. Rackert showing him the various experiments I did about the mechanism by which this enzyme worked. It was really quite interesting because this was for Rackert actually published the definitive proof paper. So he had still some questions and he read slide I would show Mitchell would say, that's consistent with the chemo is Monique theory. And Rackard would say, no, but this is why it's not right.

And so I hardly got to say a word because it's to it. I'm argued every piece of data. But you provided the substrate for the argument. Yeah, it's the data from my PhD. That was an experience. So this is early 70s? It was 1974 when I met Peter Mitchell. At then event, the next year, I decided finishing my PhD that I would look for a place that

was asking questions that were a little more biological, but I didn't want to dive too deeply into biology. So I was looking for someone who was working on like red cells or something that was pretty simple study and ended up going to Guido Guidotti's laboratory, who focused on membrane biochemistry. He was an MD PhD, but he understood medicine quite well, but his real focus was in biochemistry, was not a practicing clinician. And he was in the Department of Biochemistry and Molecular Biology at Harvard, recruited Derby Jim Watson.

And he was absolutely still a brilliant biochemist. I learned a lot from him. And he was working on sodium potassium ATPase. It had first been purified in his laboratory. The enzyme that pumps sodium out of cells and potassium into cells. And since I'd already worked on how you pump protons into cells with an ATP molecule, I thought this would be easy for me to understand. I knew how to do those kinds of assays.

So that's why I started working on it. But it turns out that Guido was also very interested in how insulin worked, because whenever you add insulin to cells, within seconds to minutes they take up a massive amount of glucose, but they also turn up the sodium potassium HPS, the sodium pump, used that sodium gradient to move amino acids into cells. And so there was a lot of membrane transport being regulated by insulin. Was it understood at that time how anabolic insulin was? What you've described are anabolic attributes, but was it understood clinically how anabolic the hormone was?

It was because, just by observation that type 1 diabetics failed to thrive. They were very small and they did not put on weight, they didn't themselves didn't grow. And so during development, insulin is an anabolic molecule. In mature adult, it no longer except under certain situations, drives growth. It will continue to drive growth of fat cells. It can also facilitate growth of muscle. But most tissues do not grow further other than those two major tissues.

So yes, it was well known, it was an antibiotic process. But now you were seeing the steps by which it could carry out that antibiotic property. Yeah, and it was generally known, what I just said was generally known. And then amino acids uptake, accelerate immediately within a minute or so, sodium exports the cell went up and glucose flux, of course, went in, particularly in muscle and fat cells, and glycogen was stored. And then of course, there was a whole lot of change in transcriptional regulation and all kinds of things.

So we knew all of that, but the actual mechanism was totally unknown. In the mid-1970s, no one had even purified the insulin receptor at no idea what the receptor was. People could quantify it because you added radioactive insulin to fat cells. It would stick to the surface and with very high affinity, and that correlated with the responses that you see. So we knew it existed, we knew roughly how many copies that were per cell, we could quantify the high rate of activity,

but no one had quite purified the enzyme to homogeneity, and so the numerous labs were trying to purify it. So eventually the enzyme receptor was purified, and it was about the same time that Sark, the Sark tyrosine kinase, the Sark oncogene, the RALS-SARCOMO virus was shown by Harold Varma's lab to be protein that had amology,

to PKA, a protein kinase, and that also was an endogenous protein that had been picked up by the virus and altered So that really was a major breakthrough. We got Nobel Prize for Harold Varmas and he shared that one with Michael right? Michael. Yeah, Michael Bishop Michael Bishop and 89. I shared 79. I think oh that they won the prize in 89 Maybe maybe I'm oh, yeah, but they won the prize in 89, maybe I'm... Oh, yeah, the prize was like...

The prize was awarded, yeah. The discovery was 78, 70. Yeah, yeah. So that was about the time the insulin receptor was being purified as well. And then Ray Erickson's labs showed that Sark had a kinase. And then Tony Harners, though, that actually by thin layer, the molecule that was thought to be

phosphatrenine was actually phosphatyracene. And phosphatyracene had never been seen as the product of a protein kinase before. It had been picked up as an intermediate and DNA unwinding, and so he could get a marker for that from Jim Wying's lab that showed that he could use on a thin layer and showed that what Sark was actually producing was actually phosphatiracing. So that opened up a whole field and then comparing the sequence of the insulin receptor to PKA versus Sark, it was clearly most highly related to Sark. And so multiple labs at that point showed that yes, the insulin receptor was like Sark, a tyrosine kinase, but in this case, a transmembrane protein had an extracellular insulin-binding

component and then the intracellular clinysectivity. So putting that in English for folks, insulin hits this trans membrane tyrosine kinase, and when the molecule of insulin hits it, inside the cell, this kinase pathway kicks up, it moves the glute-4 transporter, well, there's another step in there, but oversimplifying a little bit.

That chemical reaction is necessary to translocate this transporter across the cell to bring glucose in. The leap there, the new insight, was exactly how insulin got glucose in the cell chemically and mechanically, right? The initial observation was insulin binds. About the same time, EGF receptor was purified and FGF receptor and PDGF receptor, and they all sort of were like insulin receptor. They had an extra cellular.

And this was back, I mean, today, a young student who's in a lab doing this now is scratching their head going, what do you mean, why didn't you just use PCR or something like that? But I mean, you guys were literally what? Crystalizing a protein, or how were they actually figuring out what the structure was. So these proteins were all being purified to homogeneity by just plain fractionation. There was no way to knock out genes and knock them down. So you just had to purify the protein based on the ability to bind insulin. You just run over column after column after column and eventually you've got to a single band on a gel and they said that's your protein. And that only gives you the primary structure of the protein.

It doesn't tell you how it's folded doesn't? No, that only tells you the molecular weight basically. The only thing you get is the molecular weight. Yeah. You see it's here it is on the band. Yeah. Insulin subter is it gets cleaved but it's a heterodymer isn't it? It's a single polypeptide that gets clipped during processing in the two proteins, but are held together by disulfide bonds, so it's and then it dimer up that composition. So that we knew late 1970s and EGF receptor and all these other receptors are all look a lot like the insulin receptor. And they all had tyrosine-kines activity that was triggered by binding the growth factor insulin, IGF1 receptor also, very similar insulin receptor. One by one by one, different labs purified them.

They all found the same thing. You had the growth factor of the cell and you get the tyrosine-kines activity activated on the inside of the cell. You know, that was a huge breakthrough. But still, if you ask, when you triggered the cell with insulin or EGF or PDGF and you ask what was the major thing phosphorylated, it was the receptor itself. So if you just ask where is the radioactivity going, phosphated radioactivity, it goes mainly to the receptor. So I just like to where is the radioactivity going, positive radioactivity, it goes mainly to the receptors. I just like to mean it's autophosphorylated. So now we are left with

this several years of we have a tyrosine kinase. It explains how all these growth factors work, including insulin, and how Sark works, but we can't find anything other than autophosphorylation. How does that help? So the breakthrough ultimately then came with the observation that these autophosphorylation or phosphorylation of adapter proteins were recruiting a host of other proteins to the membrane that we're actually doing, the work that was required to drive the cell growth. And so that was, again, early 1980s.

These were all being figured out. So I got interested, as soon as I saw that there was a different kinase activity, that's tyrosine kinase, I got very excited about it, and realized there's an opportunity now if I can figure out what that tyrosine kinase does to figure out what might be the downstream signal. So we got one step into the cell. We didn't know how many steps we needed to explain glucose uptake, or amino acid uptake, etc., etc., or glycogen synthesis. So I saw a paper by

Ray Erickson's laboratory in which he found an activity associated with Sark that would phosphorylate glycerol. And he published it in JBC, it copurified with the protein through numerous steps of purification. And so, in addition to having the ability to phosphorylate, tyrosine, antibodies that got phosphorylated on tyrosine, on the purified protein, there was also an activity there that phosphorylated glycerol. And I looked at that and thought now glycerol looks like half of an anocytol

So anocytol is six carbons glycerol is three in both cases. But in the pure glycerol case It has OH on each of the carbons. Yeah, three carbons within OH on it. Yeah, and Nostal has six carbons within OH So if you took two yeah, if you took took two rings and shove them together, yeah. Loo'd them together, you would get a Nostle tall. And then what the paper showed was the KM, you know, it's a 50% concentration of glycerol, you need it for it to be phosphorylated, was something like a 100 millimolar.

Enormous concentration. Huge, like a hundred to a thousand fold higher unit ever Enormous concentration. Huge. A hundred to a thousandfold higher unit ever find in a cell. Yeah. So they weren't claiming that it was a physiologically relevant. I see. They were just saying, here's a chemical reaction that can take place.

And they just noticed it because they saw this molecule running on their thin layer that ran faster than proteins. When they isolated, it turned out to be phosphorylated glycerol. It was sort of out there. Here's, we saw this other activity, what might it be. And so I went to Ray Ericsson and said, well, that looks a lot like a nositol, so why don't we... It was Ray at Harvard at the time?

Yeah, one floor below me at Harvard. So my graduate student, Malcolm Whitman, who knew how to do kines assays, and small molecules collaborated with Ray Erickson's post doc to see whether phosphatinal anositol might be a better substrate than glycerol. And he tried it, and sure enough, he got this KM now of like five micromolar. Wait, wait, wait, wait, how is this even possible? Let me make sure I understand what you just said.

You take glycerol and you need a KM of 100 millimolar to get it phosphorylated. You simply take the same structure, but now it's basically two of them stuck together in a ring and you get down to five micromolar. It's also on a membrane component, so the inocytol is in a membrane bilayer. So you sonnicate the lipid, and instead of free molecule floating around. Oh, so it's not a free inocytol. It's the inocytol in the phospholipid,

or in the lipid. Yeah. Tell me from a chemistry standpoint, and I apologize, I know that for some of you listening right now, you're thinking, wow, you guys are really in the weeds. I promise we're gonna get out of the weeds in a minute, but I also think this is just an interesting example

of the specificity of biology, too. What is it about that lipid holding that, is it the position with which it holds the ring in place that enables that phosphorylation? Well, now that we know the structure of PI-3 kinase and how it works and its mechanism, it's easy to explain and retrospect. But at that time, we didn't know that there was anything there other than Sark. It was hard to explain why the Sark, Tarzan Kaini's itself, was carrying out this reaction. What did the editors even say of that? I mean, that's one of those things where people are like, is there a mistake here? Because that's like a five-log difference. Physicists had no problem understanding what I just said.

If you combine things to two dimensions, they can come together much more readily than if you do it in three dimensions. And that would account for the difference? Yeah, easily. Try closing your eyes, bringing your two fingers together in space and three-dimensional space, and then do it again on the table. When they're on the table, yeah. You'll find it. Yeah. Yeah. So, combining things to two dimension is way nature continually uses Membranes and the lipids basically keeping it in a two-dimensional plane. Yep. Yeah, so an event We're really getting in the weeds or not Just personally I I mean all of that is intuitive to me

What's not intuitive to me is five log difference? I would take a one log difference for what you said That's what's amazing to me. I didn't. Well, there are other reasons that will come clear. Let me move on. So the bottom line was that that Sark preparation, which we assumed was completely pure, wasn't completely pure. It was a certain amount of the Sark harvested out of the cell, brought along the second enzyme, which, let me turn it out to be PI3K, phosphonositide 3 kinase. But we didn't know that at that time, and so we suggested in the paper that the Sark, very same enzymatic

pocket could both accommodate tyrosinus as substrate, and also accommodate the head group of phosphatotol anacetol. That in the end turned out to be incorrect, even though by all criteria that we could characterize at that point, we couldn't prove that there was a separate function. If you used a kinase dead shark, or a mutant of shark that no longer had activity, then the phosphatotol anastotol kinase also went away. That's why we thought it was the same pocket that was doing both. Retrospect, it turns out that the enzyme has to be active in order to bind the PI3 kinase, and that's why that activity was coming along. So we published that. We're very, very clear to say that we used sonicated membranes to do the assay, which was necessary to get this

many log preference for that substrate. In the meantime, after publishing that, multiple other labs tried to reproduce it. But they didn't have a machine that would sonify the lipids to make the membrane by layers. So they just added detergent. So even though we showed in our paper that if you added detergent, the enzyme activity completely disappeared. Wouldn't the detergent break the glycerol off the lipid? The phosphatinal inocetol would now get embedded into the detergent rather than being a bilayer. Yeah, it wouldn't stick to it at all, right? Right.

So you're going to lose that advantage of having a membrane. And that wasn't known at the time, I guess? Well, we knew it because I'd worked on membranes for the previous 10 years. But the other labs didn't, I guess at the time they didn't know that. They were not membrane biochemists. I get it all, molecular biologists. So they tried to reproduce the result, but they didn't have a sonifier to make membrane lipids, and they assumed a little bit phosphorylates, the hechrofytin, phosphatonic

and acetol, it shouldn't make any difference how you present it. And so they presented it to the surgeonll and they could not reproduce the results. So there were three prominent papers coming out a year or so later. Saying, no, this is incorrect, this is incorrect, this is incorrect. From major laboratories. So at that point, it already started collaboration with Tom Roberts. But at that point, Lou, did you have confidence that you had done this correctly or were you questioning yourself?

I had no doubt. So you knew this was a methodologic error on the parties of the three labs, and despite the fact that at this point, the scientific community would look at you and say, Lou, you're probably wrong. You were confident in your methodology. Yeah, there was no doubt. All my research had been on membrane enzymes and membrane reconstitution, etc. So I knew what I was doing made sense and I knew our results are reproducible because not only could multiple people in my laboratory do it, the multiple

people in Tom Roberts laboratory who we started a collaboration with could do it and Brian Schaffelson's lab, which was a third collaborator, could also reproduce it. So multiple people in multiple independent labs all got the same result. If you solidified the lipid, and if you added MP40, a detergent to solidify the lipid, then the activity went away.

So there was no doubt about that. And so I had no lack of confidence that we were right, but the problem was how to convince these other laboratories that they needed to do the essay right. They didn't want to buy a son of fire. They were like, I'm done with that when there didn't, it doesn't work. So we actually sent David Kaplan, the graduate student Tom Roberts lab in Malcolm Whitman, the graduate student of my laboratory, to these other laboratories with the son of fire. They went there, they sonified the lipid

form, gave them to their their graduate students who then did the essay again, and now they got the same result we got. Are you pushing this hard to get through the labs to see it because you know how important this is going to be or you have sort of premonition about the role that PI3K is going to play in growth. I mean, you couldn't have possibly seen in the early 80s what you know today. So how much of this was just scratching an intellectual itch versus a biologic intuition about the importance of this to overall growth, which is obviously where we're going.

I'm amazed we are this far in and we haven't used the C word yet. So the reason I was completely convinced by 76, 77 that this was driving growth of cancer cells is that the cloverage we started with Tom Roberts' lab. And Tom Roberts had been working on polyoma midalty. So polyoma virus is a DNA virus, while Sark is an RNA virus. So it causes, as the name implies, the formation of multiple tumors in mice.

So you infect the mouse with this virus and all kinds of tumors show up everywhere. And so, Tom had began mutating various regions of polyamimital T. And polyamimital T, the reason we looked at polyamimital T, is because his lab, and so our courtines' lab, and others, and shathausen, had shown that Sark coped purifies with polyamimital T.

We are already new by the mid-70s that Sark was somehow implicated in how polyamomital T transform cells and forms all these tumors. That's why I went to collaborate with Tom because he had additional tools that we could collaborate with to understand. And so he had made all kinds of mutations. He'd also found that Sark phosphorylated site, then Polyamomital T, a tyrosine site, was highly phosphorylated. And even more importantly, a tyrosine site was highly phosphorylated, and even more importantly, that if you mutated that tyrosine residue to phenylalanine, polymer-middle T completely lost its ability to transform cells. But a single point mutation.

Even though the Sark protein was still bound, there was no longer tyrosine phosphorylation 315 and that eliminated the ability to transform cells. So we said, well, if pi3 kinase is not sark itself, maybe it binds to middle T independently. But we found that if you prevented sark from binding middle T, then you didn't get that tyrosine phosphorylation. And now there was no pi3 kinase activity bound to middle T. So you needed sark for middle T to bind to PI3 kinase. At this time we didn't know it was phospholid in the three positions, which we'll come back to. However, the shocking result was that if you eliminated that tyrosine residue,

now PI3 kinase would no longer bind, and metal T would no longer transform cells, even though Sark was still there and was still activated. So that said that activating Sark is not sufficient to transform a cell, unless you also activate PI3 kinase. And we probably said in nature in 76, I think 76, 77 in nature. Were you collaborating with Bishop and Varma?

I'm sorry. 86, 86, 87. And then 86, 87. So 84 was when we published the paper with Ray Ericsson, with Bishop and Varma's point. I'm sorry. 86. Okay, okay. And then 86.87. So, 84 was when we published the paper with Ray Erickson, and then a couple years later with Tom Roberts, and a couple of papers we had. And we showed that PDGF receptor also when activated, brought down the copres of the PI3, kind of, sective, et cetera, et cetera.

So that's where we were in 1986, 87 or so. And at least some of the people who had gotten sonnacators also believed us that this was really uniquely associated with tyrosine kinases after they were activated by growth factors. We saw the insulin receptor would also bring down PI-3 kinases only if used to make insulin first.

Then you could also bring down this PI kinase only if you use stimuli with insulin first, then you could also bring down this PI kinase activity. But at that time, the only flano phospholid form of phosphatotol Nostal known was phosphatotol Nostal for phosphate. That was discovered in 1949, I mentioned earlier. There's a four phosphorylation and there's a four plus five phosphorylation. And those are the only two species of phosphorylated phosphatonal enocetal. So we assumed that this was phosphorylation at the four position. But as we began to characterize the ability of proteins from cells to phosphorylated phosphatonal enocetal, we found that there were two activities.

There was one that required that usonic thylamin membranes for it to bind and get for it to have activity. And another that would work perfectly well, in fact, even better, if the lipid was dissolved in a detergent. So we called those two enzymes type one and type two, PI kinase. And it was only the type one and type two PI kinase. And it was only the type one,

the one that required sonified lipids that co-precipitated with all these tyrosine kinases. The type two had completely different enzymatic characters. So it was inhibited by a denison, the type one was an end. So we had a whole lot of profiles that said these were two different enzymes. So Malcolm Whitman, registered in in my lab decided to separate them. So we ran column fractionations and metadiseous activities with or without detergent, with or without adenosine, and he characterized

the two and some separated them completely. And he had a lab, we had a lab meeting which every other spot on the thin layer, which is how we characterize the lipophosphorylation, we ran it out to separate the molecules based on migration in a solvent on a silica plate. And we noticed in the lab meeting that every other spot migrated about one millimeter different from the previous spot. And the way that Markman spotted them on the thin layer was it was type one and type two, then type one,

and type two, type one, type two, all the way across. If you looked at where the radioactivity ran, it went up, down, up, down, up, down, up, down. And the bottom line was, to me, as a chemist, the fact that they migrate differently means they have to be chemically different. That meant that one of those molecules was being phosphorylated at a different position on the enocetal ring from the other. So we began the process, then, of chemically characterizing the product of the type I and the type II enzyme.

And how subtle was this, by the way? Was this obvious in looking at it? Or was it something that could have easily been dismissed? I guarantee almost everybody would have dismissed this. One millimeter. Yeah, I was about to say that just doesn't sound like... The spot was at least 10 millimeters diameter in the center of this spot, so we're one millimeter different. Do you naturally have an eye that gravitates towards symmetry?

I have an eye that gravitates towards unexpected results. And to me, there's no way to explain it. If you had only two spots beside each other, you could say, well, that side of the thin layer migrated a little faster because of the solvent front was not completely horizontal. But this, you couldn't explain the up, down, up, down, up, down all the way across. Do you still have a photo of that? It's in figure one in the Nature Paper that we published. From 80, 7, 88.

88. Probably say 88. We'll link to that figure. But the Obseration was made in 1987. I bumped into Peter Downs, who's one of the best lipid chemists in the world, and at a meeting at Coltspring Harbor, and I told him this result, and I said, I know you must have standards for a Nostatal degradation. So, can we work together?

And so, we knew it had to be some site other than the four position, but it could have been the five, it could have been the three. Yeah, was there a chance it could have been you were looking at four versus four plus five, four versus four plus five? No, because we knew at four plus five, it would migrate to about three inches different. So that would look totally different. So the null hypothesis is you're just looking at four and your assays a little dirty, but you're thinking, no, it's something else.

Yep. Of course, we repeated it multiple times, and we also did HPLC separation, reverse phase, every way you could look at it, and in every way we did it, they were chemically different. They migrated differently and numerous. The nature people we only put that one slide, that one figure of the slightly different migration. Because from there on, we did the chemistry to prove that it was a three position. Why do you think the three position had such a similar look to the four versus the five? Well, keep in mind, there were only two species known to exist. At the time, we made this discovery, phosphatodonocytol, four phosphate.

Oh, and four five. And four five. It was not a five by itself. It was not a five by itself. Got it. and we made this discovery, phosphatodonacetol 4-phosphate. Oh, and 4-5. And 4-5. It was not a 5 by itself. It was not a 5 by itself. Got it. So this told us there's a 3 by itself. And then we went on to show there's also 3-4,

and also 3-4-5. PI3 counties could phosphorylate the 3-position, whether or not the 4 or five were already phosphorylated. So we now that generated three new species, PI3P, PI34, and PI345. But I have to say the 345, this is sort of another sidebar that we could drop out of this, but it's so cool, never published it, because it was embarrassing, but you know, at this age it may as well get embarrassed. So we knew that there were species that had been claimed to be four-five, that were probably

three-four, for the same reason that the three-p was thought to be four-p, because separating the three-four from the 4.5 was also hard. So we looked for that carefully and found that in fact there was a 3.4 being made and proved that the same enzyme was making 3.p. and if you gave it p.r. 4.p. to substrate, it would make 3.4.p. too. Let's leave seroonion and a post-doc in the laboratory was doing the experiments to really verify that those two species really were being made from the same enzyme.doc in the laboratory was doing the experiments to really verify that those two species really were being made from the same enzyme. And in the course of that work, she came into my office

one day and said, I no longer get the same results that I've been getting for the last six months. Something has changed. And so we went over all of her thin layers. Again, this is how we separated all the species. And sure enough, after getting numerous results over all of her thin layers. Again, this is how we separated all the species. And sure enough, after getting numerous results in which she could give PI4P and get PI34P2, suddenly over the past week, she no longer got that doubly phosphorylated lipid when she

added purified PI3 kinase to that liquid. So the question was what had changed. And she said, well, the only thing that changed is we ran out of the PI4P and I had to buy a new jar of PI4P from Baron Germanheim. And I said, oh, that's interesting. As I looked at the thin layer more carefully, I noticed that just when the experiments were no longer working, there was a new spot on this in layer that was just off the origin, where we assumed it would probably just be ATP.

There was always a little bit of contaminating ATP that got left over when you separate the lipid out. And we assumed that that was ATP. But I noticed it ran just a little bit faster than ATP, and that it only appeared in the experiment where she could not find the 3, 4, and I said, okay, stained is what I had, and it will tell you where the lipid that you used from barren germine runs. And I'll bet that it's actually 4,5p2. And that turned out to be the case. That they'd mislabeled the bottle. What was commercially sold is phosphatoidal enocetol 4-phosphate,

was actually phosphatoidal enocetol 4-5 bisphosphate. And so by accident, I almost think that God looked down on us and said, look, they're missing the most important lipid PI 345 P3. It never occurred to us to look for it because we were no one had ever claimed. There was something like that that ran on a thin layer. That was because it runs so close to the HEP that was always missed. Because it's got three phosphates. Yeah, it's very highly charged and therefore runs. I never once thought of that until you just said that, Lou, I never I never would have had that thought actually. And then of course when you say it now, I mean, that's think about how dominant those three phosphates are, right?

In fact, a year after we made this observation, the paper came out. In fact, I got called by nature, saying there's this paper claiming they found something that looks like triply phosphorylated phosphatotoninocetol. And they asked, do you think that's possible? And I said, yeah, in fact, I know it's possible. We already have results showing that that can happen, which purified enzyme. Well, thank you so much for the generosity with your time and your insights and most importantly for the work you're doing, you and your colleagues are helping a lot of people.

Thank you, Peter. Thank you for listening to this week's episode of The Drive. If you're interested in diving deeper into any topics we discuss, we've created a membership program that allows us to bring you more in-depth, exclusive content without relying on paid ads. It's our goal to ensure members get back much more than the price of this subscription. Now, to that end, membership benefits include a bunch of things.

One, totally kick ass comprehensive podcast show notes, the detail every topic paper person thing we discuss on each episode. The word on the street is nobody's show notes rival these. Monthly AMA episodes are asking me anything episodes, hearing these episodes completely. Access to our private podcast feed that allows you to hear everything without having to listen to spills like this. The Qualies, which are a super short podcast, typically less than five minutes

that we release every Tuesday through Friday, highlighting the best questions topics and tactics discussed on previous episodes of the drive. This is a great way to catch up on previous episodes without having to go back and necessarily listen to everyone. Steep discounts on products that I believe in, but for which I'm not getting paid to endorse, and a whole bunch of other benefits that we continue to trickle in as time goes on. If you want to learn more and access these member-only benefits, you can head over to peteratia-md.com forward slash subscribe.

You can find me on Twitter, Instagram, Facebook, all with the ID, Peter Atia MD. You can also leave us a review on Apple Podcasts or whatever podcast player you listen on. This podcast is for general informational purposes only. It does not constitute the practice of medicine, nursing, or other professional health care services, including the giving of medical advice. No doctor-patient relationship is formed. The use of this information and the materials

linked to this podcast is at the user's own risk. The content on this podcast is not intended to be a substitute for professional medical advice, diagnosis, or treatment. Users should not disregard or delay in obtaining medical advice from any medical condition they have, and they should seek the assistance of their healthcare professionals for any such conditions. Finally, I take conflicts of interest very seriously. For all of my disclosures and the companies I invest in or advise, please

visit peteratiamd.com forward slash about where I keep an up-to-date and active list of such companies. you you