#216 - Metabolomics, NAD+, and cancer metabolism | Josh Rabinowitz, M.D., Ph.D.

Hey everyone. Welcome to the Drive Podcast. I'm your host Peter Attia. This podcast, my website and my weekly newsletter all focus on the goal of translating the science of longevity into something accessible for everyone. Our goal is to provide the best content in health and wellness, full stop, and we've assembled a great team of analysts to make this happen. If you enjoy this podcast, we've created a membership program that brings you far more in depth content. If you want to take your knowledge of this space to the next level, at the end of this episode I'll explain what those benefits are. Or if you want to learn more now, head over to peterattiamd.com subscribe Now without further delay, here's today's episode. My guest this week is Josh Rabinowitz. Josh is a Professor of Chemistry and Integrative Genomics at Princeton University, where his research focuses on a quantitative, comprehensive understanding of cell metabolism through the study of metabolites and their fluxes. He's also the Director of the Princeton Branch of the Ludwig Institute for Cancer Research and a member of the Rutgers Cancer Institute. Josh earned his MD and PhD in biophysics from Stanford, which is how we met. We were in the same graduating class, although he of course started earlier because he did two degrees. In between earning his MD and PhD and joining the faculty at Princeton, Josh worked at Alexa Pharmaceuticals as the co founder and Vice President of Research, a topic that we actually touch on in this podcast. Josh is the inventor of over 160 patents, including five drug products that are in the FDA sanctioned clinical testing pipeline. He has received numerous awards including an NSF Career Award, an NIH Pioneer Award, and was distinguished as an Allen distinguished investigator in 2019. This is a pretty technical episode, I'm not gonna lie, and we really focus on three things. Metabolomics, NAD specifically and of all of its sort of precursors and movements, and cancer metabolism. We open the discussion talking about metabolism, metabolomics and fluxomics and this includes a pretty in depth conversation around glucose, glucose metabolism, lactate as a fuel, movement of lactate, and the regulation of these substrates. From there we speak in more detail on the electron transport chain and the Krebs cycle and what the implications are both with respect to drugs and nutrition. This is an important segue then into the second major pillar of our discussion which is that around nad. Most of you have heard of nad. We certainly get a lot of questions about NAD and not so much about NAD as we do probably more about their precursors, NR and nmn. We've also had previous podcasts where We've discussed this, including episodes with David Sinclair and Rich Miller. So in this discussion, we talk about the intravenous use of nad, the oral use of the precursors, and I'll just give you a little spoiler alert, or I'll try not to spoiler alert it, but I'll point you to something which is I learned something pretty significant in this episode that I have historically been saying incorrectly for some time. So if this is a topic that's interesting to you and you've heard me speak on it before, you might want to listen to this because I'm going to come into a big correction. We end our conversation talking about cancer metabolism and particularly one way in which cancer metabolism and immunotherapy might intersect. So without further delay, please enjoy my conversation with Josh Rabinowitz. Hey, Josh, great to see you again. It was about three years ago, I think was the last time we saw each other in person at some sort of conference in New York, a cancer metabolism conference, I think. Right.

The New York Academy of Sciences, I think.

And we somehow wound up at some kind of mediocre bar in Brooklyn. And it might have been the dorkiest concentration of people talking about autophagy and metabolomics and all sorts of things there was.

That was actually the AACR Brooklyn conference. That's true.

Well, you know, it's really funny. Just recently, I've interviewed a few of our classmates from med school, Max Dean and Carl DeSeroth. And I think Carl and I were reminiscing about how you, me and Carl all started our surgical rotation together in the same day. It's almost 25 years ago, and I remember it like it was yesterday when we were all sitting in the room practicing sewing with our big goofy knots and things like that before we all got divided up into our surgical rotations. Do you remember that?

I definitely remember you. I really do not remember Carl from those days, but I certainly remember you practicing a lot while I looked and said, oh, I guess that's what you're supposed to be doing if you want to become a surgeon. The thing I remember most clearly is being in surgery with you. And it was like the very end of surgery rotation, finally being given the Bovie, immediately making a mistake. Then I got the nice lecture. We're happy to pass you on this rotation as long as you promise never to use a knife or Bovie again.

Remind me, whose lab did you do your PhD in?

I did my PhD with Hardin McConnell, one of the great physical chemists, together with Mark Davis, the immunologist Tell me.

And tell the listeners a little bit about what project you worked on for your thesis.

My thesis was about the physical chemistry of T cell activation. At that point in time, people had discovered that different antigens could activate T cells differentially. And so I was really interested in how that happened. And so I studied the process of antigens turning into peptides that could bind to mhc, and then how the kinetics of the interaction between the peptide antigen and the matrihistic compatibility complex protein mhc, and then the interaction of that complex with the T cell receptor determined whether people have productive or failed immune responses with the hopes you could then manipulate those processes to promote better vaccination or disease clearance and also to potentially treat autoimmunity.

And did you also have an interest at all in cancer? Because, of course, this would be one of the hallmarks of how immunotherapy would be effective in eradicating cancer.

Shows how bad a prognosticator I am. At that point, I really felt immunological approaches to cancer didn't hold that much promise. And so I was really more focused on infectious disease and autoimmunity. Shows you what I know. It's wonderful to see that the world has turned out to be a lot better than I dreamed it would be on that dimension.

It's such an interesting topic. I had Steve Rosenberg on the podcast last year, and it was a beautiful and fun recap of how the immune system works in general. But we were talking about it, obviously through the lens of cancer. And I think the part that will forever humble anyone who tries to think about how amazing the system is is that these things have to be 9 to 11amino acids long. I mean, the peptides have to be just the right size to. To be presented and then to be recognized. And that just doesn't seem to leave a lot of margin for error. I mean, it is really a tuned system. Do you have a sense of why evolution ended up with such a narrow fragment of peptides that were recognizable, as opposed to a broader range, or as opposed to just a range that's different? Like, why wasn't it 2 to 3 or 150 to 160amino acids? Like, is there a chemical reason? Because of your background in chemistry, I feel like you'd be more equipped to offer a teleologic explanation for this.

I do have an intuitive sense on this, that our bodies work on the scale of billions of immune cells and billions of immune receptors that are made through recombination and that naturally pairs with billions of antigens. And so you just think about this number nine and you think about the number of amino acids, right? There are 20amino acids, so you're talking about 20 to the ninth power of presentable peptide antigens. And so these things are all tuned to be on the same scale, this kind of scale of billions and tens.

Of billions in that case.

Exactly.

If it was like a three peptide, it's really a small number, actually.

There's not enough information there to selectively respond to a virus or a bacteria.

And if it was 25 peptides, it's too much. There's too much information there.

There's extra information anyway. And this system, I think was built to work on minimal or just the right amount of information.

Yeah, that's super interesting. So at what point during either your PhD or the end of medical school where we met up in the clinical portion, did you make the decision that you wanted to be a full time scientist as opposed to a physician scientist?

I applied to do an internship knowing that I really loved research. But I guess I decided as I did more and more medicine, that medicine is such a noble profession, but it involves a lot of doing the same thing right over and over again, a lot of following the standard of care, even for the most creative physicians. And that ultimately my passion is to come in and try to do something different every day, to think differently than people ever have before. And so that's what really led me to research. I love the patient interaction part of medicine. It was the doing things right part that was challenging for me.

Sometimes I think those of us that chose the more medical side of things can also speak to the frustration of how creativity can often be stifled in medicine. And that's in large part for good reason. But I think it comes at a cost as well. I think, I'm sure. On this podcast I've told stories about how frustrating that was in surgery. At least surgery, probably more so than most other disciplines, tends to sometimes at least frown upon creativity and novel approaches to problem solving. If I remember correctly, before you joined the faculty at Princeton, where you are now, you went into industry straight from medical school. Am I getting my facts right?

I was fortunate to have the opportunity to work with one of the great early biotech entrepreneurs, Alex Zaffaroni. He and I started a company when I was straight out of medical school that was focused on fast drug delivery. So what could you do by being able to deliver medications non invasively on the timescale of giving an IV push in the hospital? We did that through inhalation of small molecules. Kind of building on the concept, obviously, that if you smoke something like a cigarette, you get incredibly rapid access to the systemic circulation. We built that company. Lexa Pharmaceuticals, still exists, has one FDA approved drug. That was my first job.

What drugs were you targeting with that?

You know, our initial focus was migraine. Unfortunately, we never found the drug that had the perfect combination of safety with rapid delivery and efficacy for migraine. The drug that ultimately got approved was for acute agitation, the sedative hypnotic for acute agitation. And I'd say it's really wonderful for the patients who get it. They come in the er. It's something people don't always know about agitated patients. They're agitated, they're frustrated, but they also want to stop being agitated. They don't want to act like that. And so they're very eager in most cases to take a puff of something and calm down in a couple minutes. It's wonderful for them.

So this is kind of an unusual path. I'm guessing that most people who experience that type of success that you had would want to keep doing it over and over again. I mean, hence the term serial entrepreneur. What made you decide to take this lateral step to go into academics, which would have made a lot more sense if you'd done it coming straight out of your PhD.

You know, I think I was lucky to get the chance to go straight from that job into a faculty position at Princeton. That's a rare opportunity to have because.

You didn't do a postdoc in there unless they considered your industry as sort of a grandfathered postdoc.

Yeah, it was a very, very weird version of a postdoc. It united me with my wonderful wife at Princeton on the faculty, and turned out well.

So you show up at Princeton and they put you on a tenure track position, which means they're giving you the types of resources to now start solving whatever problem you want. What was the first problem that you said I want to build a lab around?

You know, one thing that I learned actually doing Alexa was about drugs, because we studied every medication in the pharmacopoeia for whether it could be a candidate for rapid delivery. Could you make a benefit by delivering it rapidly? And one thing I noticed is that so many of the most important medications work via metabolism. Then starting out my lab, I realized that there were relatively few labs looking at metabolism broadly compared to other really important areas of science, like immunology or cancer or neuroscience. And so I started my lab with a really simple question. Could we measure the classic metabolites that you read about in a biochemistry textbook in one shot, quantitatively. And so that was the starting point for my lab. And I guess the second question that we always had in our mind is, can we measure the activities of those metabolic pathways? So how fast are those metabolites flowing? Where are they coming from and where are they going?

I had lunch or dinner actually, with one of our mutual friends, Navdeep Chandel, who was also a previous guest on the podcast three or four years ago, maybe a week ago, and he was here in Austin for a talk. And we were talking about how in the late 90s, he said, and he was obviously studying metabolism, he said, if you were to rank me, meaning Nav was ranking himself. Right. If he said you were to rank me and my work, just across the spectrum of the stuff scientists were doing, he said, I'm bottom 10 percentile. Nobody found metabolism interesting. This was. I forget the term he used, but, like, this was basically the corner where the kids went that nobody wanted to play with. If you weren't doing genomics, if you weren't doing this other sexy stuff, immunology, you were really an uninteresting person. But he just found it interesting. And of course, today, as we're going to discover, this is where the action is. You know, Nav was talking about that in the 90s, we're now in the early 2000s. Was it still a little bit of that stigma that Nav described, where he was like a totally underperforming loser in his own words? Or was that transition already starting to happen where people saw, wait a minute, there's something going on here.

Nav is so funny, and I don't think anyone who's ever met Nov thinks he's a loser. I'll say that right off the bat. First, I will say that metabolism as an area was definitely out of favor. And it was a really strange thing because you had the academic current that metabolism was a solved problem. Krebs was the culmination of metabolism research at the same time that metabolic syndrome was becoming worse and worse and worse in the population. When I started, it was still not a real popular topic, metabolism. But I think two things were beginning to shift. One is the fact that people realized that genomics as a standalone was not going to solve health problems, that it was really going to have to be supplemented by other technologies that looked at biochemistry broadly. And metabolomics proved to be one of those been enduring and will be enduring. And the second is that the metabolic syndrome epidemic just kept becoming more and more obvious.

So this is kind of where your training as a physician becomes relevant, because perhaps more so than somebody who didn't spend four years also doing medical school, you saw the clinical problem that was sort of kicking you in the face, even if it wasn't top of mind to a scientist.

I think it's really true. I just benefited so much from the breadth of biology and medicine that you learn in medical school. And, I mean, it allowed me to start my lab working on bacteria, which I had never worked on at all as a PhD student, because you learn bacteriology is one of the things in medical school. And that was like the perfect starting point for building these technologies with a view all the way to metabolic syndrome and ischemia, reperfusion injury. Right. These huge medical problems. But I really wanted to start somewhere tractable where we could get firm proof of concept.

So for somebody listening to this, let's assume that they have a greater attention span than somebody you're going to walk into at a cocktail party. But obviously not necessarily the depth of understanding of everything we're about to go into. How would you explain metabolism to that person?

Metabolism is the process that converts the food we eat into usable energy and the building blocks our body needs to grow or regenerate itself, as well as waste along the way.

And so now how do you layer on the omic piece of that, which is really what we're starting to talk about. When people hear the word genomics, they sort of understand what that means. But I think when people hear the term metabolomics, it becomes a little harder to understand. So how would you now layer in that, in the context of everything that you're now beginning in your lab, the.

Bulk of activity and metabolism that makes most of our usable energy involves something like 100 metabolites. And so the first thing we really wanted to be able to do was measure those hundred metabolites really well, tell.

People some examples of those. Josh, we take it for granted, but, like glucose would be an example of a metabolite. What are some other metabolites that are important to understand if you're trying to study this system?

All the amino acids are other fundamental inputs that we get from the diet. Like glutamine is a great example. That's a very important circulating nutrient. Other things that come in from the diet, if you have vinegar, you have acetate, or else your microbiome can make acetate that goes in the body fats are obviously important input metabolites. And then there are sets of intermediary Metabolites, these are things that are like glycolytic intermediates that people may have heard about in biochemistry. Fructose bisphosphate is a famous one of those pyruvate lactate that a lot of people hear about from exercise, members of the Krebs cycle, like citrate is a famous one that exists in our circulation, obviously in citrus fruits. So these are classic examples of metabolites. And then there are the more effector or energy holding metabolites like ATP, nadh, nadph.

So I kind of interrupted you there, but you were kind of explaining how first you begin with sort of this survey of all of these metabolites.

We want to be able to measure all of these. These are kind of the core components of metabolomics. And part of the beauty of metabolism as a system is that with some modest variation, there's almost a singular solution on earth to how metabolism works. And so when we learn to measure the metabolites in E. Coli, at the same time, we were really learning how to measure metabolites all the way up to human. There are these basic components of protein and nucleic acids, basic intermediates like fructose bisphosphate that exist at all of these levels. And if you survey relatively comprehensively, there's on the order of a thousand of them that have clear biological function. So it's a big problem, but it's a problem on the scale of knowing all the kids who go to your high school and not a problem of knowing everyone in the phone book in New York City. Right. So it's a problem that's right at this interface between the human scale and the computational scale.

So I wanted to ask you about that. You already kind of anticipated perhaps almost a question which is, do we think we have the complete solution set here? I mean, we clearly know all of the amino acids, we clearly know all of the intermediate steps and intermediaries, period of the Krebs cycle. Do we think that that means we actually know every single metabolomic element? Or is there a chance that there are others out there that we don't know because we haven't looked for them? Or they're very short lived, for example, and we haven't stopped and looked at reactions closely enough or studied the kinetics hard enough? This is almost a naive question in a way, but I've never actually thought about it until now.

It's a great question. And we keep discovering new metabolites. Groups around the world keep discovering new metabolites. I would say it's a Interesting yin and yang because there's this steady accumulation of new metabolites. But I don't really think there's been a completely new and obviously important metabolite yet this century. At some level, in terms of the metabolite finding part of the problem, things were wrapping up around the time of Krebs, the most important part of the work anyway. But in terms of undertoning how the system really works and how we can choose the right diet to be healthy, given our genotype, given the disease we're fighting, we haven't scratched the surface yet.

How many of these metabolites are really tightly regulated a la glucose versus not that regulated at all. They can kind of fall to zero and they can, meaning how many of these things can change by log orders all over the place? And how many are regulated so tightly that if you just fall a little bit out of that range? One of the things I try to explain to people when I explain regulation and homeostasis is I love using ph as an example, because, you know, the ph spectrum runs from basically 0 to 14, neutral being 7. But anybody who's taken care of a patient in the hospital knows 7.4 is where we live as an organism. Almost unsurvivable to have an acidosis that goes below 7 or an alkalosis that exceeds about 7.7. So for a system that runs basically 0 to 14, the fact that we can't as a species survive outside of 7 to 7.6 or 7.7 talks about something that is so tightly regulated. So in that field of metabolomics, which ones behave like ph and which ones don't?

Ph is such a great example, right? You have this giant logarithmic scale from 0 to 14, right? And so even when you talk about 7.1 to 7.4, you're talking about something of a two, three fold change.

It's about a two to three fold change in acid concentration.

A lot of metabolites, the important ones, live typically in that two to three fold range as being the preferred range. Some of them there's a lot more active regulation like glucose. Some of them there's kind of relatively passive processes that tend to keep them in that range. Then of course, there are all sorts of other metabolites that may be some cool secondary metabolite that's made by a plant. And some of us eat it, some of us don't eat that plant. And so some of us may have a lot, so some may have none. But for the big ones, the biochemistry textbook ones, this Kind of fewfold range in the bloodstream. Is common, healthy place to be?

Are there common and consistent tools that the body uses to regulate? Are there principles that the body just adopts over and over and over again in the form of this regulation?

There is one most important principle, and that's when it's there. Use it up. In a physics speak, you could say this is a linear consumption of circulating metabolites. Or in chemistry speak, people call this mass action. Just whatever mass is there, you tend to take it in. A lot of what you eat after a little bit of processing enters the bloodstream, and then it's the job of tissues that need these ingredients to use them and use them at first blush, in proportion to their availability in the blood.

Are there examples where that regulatory mechanism is not the preferred way to manage them?

Well, there's a lot of regulation layered over top of that in order to make the body work. The most important regulatory hormone in mammals, I'm pretty convinced, is insulin. You know, there are two ways to look at insulin, I think, and there's probably the way that comes to mind first to you, which is insulin is a hormone that acts to control elevations in blood sugar, and it does that at the highest level by promoting uptake of glucose and preventing production of glucose. But there's an alternative way to look at insulin, and that's that we've evolved mainly to be able to survive lack of nutrients, that this was the strong selective pressure on animals and mammals, and that storing fat is very precious and that insulin is a hormone that says you don't have to use fat right now. Okay. And so it senses that there's enough carbohydrate around and therefore it's safe to not release free fat from your adipose tissue.

Does that mean that you think that an equally important role of insulin is not just the disposal of glucose into muscle and the cessation of glucose production in the liver, but you're saying it's equally important as a signal to stop lipolysis, to keep your fat in its fat stores. Meaning, save this for a rainier day, because you actually have the glucose here that I'm going to deal with.

That's certainly how I look at insulin right now. There's little doubt biochemically and ethically, the suppression of lipolysis is a primary, perhaps the primary function of insulin.

Going back to the broad strokes of metabolomics, you alluded to it briefly, without I think, using the term. But what do we know about the flux of these things? When I think back to even my biochemistry 25 years ago. Stryer's textbook, which is the classic textbook, at least it was then, I imagine it's one of them still today. We really studied it in a static way way. And I'm guessing that that's one of the first questions you went after. Right, which is what's the movement, what are the derivatives with respect to time of all of these things. Maybe expand a little bit on this idea of fluxomics.

Metabolism is a system in action. And I think this kind of static view of metabolism, which is probably never a view that Streyer ever had in his mind, but that got codified in the textbooks, is one that killed metabolism in a way, as a topic of excitement. Metabolites are intermediates in the process of converting what we eat into usable energy and protein biomass. And these things, they're really relatively low in abundance and they're flowing very, very fast. So they're completely different than parts of our body, like neurons that are going to sit there maybe for our entire lifetime here. The metabolites are meant to be made and used somewhere on the timescale, depending on the metabolite of roughly a second to roughly an hour, maybe for metabolites in the bloodstream. And all the action is in the flow. It's really understanding where things are coming from, where they're going, where we can learn about how metabolism works.

Let's again just use glucose. Because one, it's ubiquitous, everybody gets it. It's essential for life. But it also offers, I think, a beautiful portrait in velocity. I just had my blood drawn yesterday. I draw my blood about every two months and tube of blood comes out. And let's say my glucose, because that's a snapshot, right? That's literally in that moment. Out comes five tubes of blood and it's gonna look for a whole bunch of things, but one of them is glucose. And my glucose, cause I did a finger prick at the same time. My glucose was 89 milligrams per deciliter. Can you explain to people what that actually means? What does it mean that my glucose at that moment in time was 89 milligrams per deciliter?

Well, I'm sure you were smiling about it. It's a super healthy blood glucose in terms of the level. But when you think of that absolute amount of glucose, right. If you took all the glucose in your bloodstream at that level, that's a few minutes of glucose or energy.

Yeah. It's probably what, 4 or 5 grams of total glucose, 20 calories. Worth.

Exactly. So that has to be constantly replenished in order to feed your brain and the other tissues, like activated immune cells that depend on glucose.

Glucose. Now, this is what to me is remarkable. If I had done that same test, Josh and I had come back, let's make the math easy and say it was 90. We'd still say, great, you're healthy, your fasting glucose is 90. Now, let's say I had come back and it was 180 milligrams per deciliter, my fasting glucose. There's a disease that I would immediately now know that I have. That disease is called type 2 diabetes. What's the absolute difference in the amount of glucose in my bloodstream? It went from being 5 grams to 10 grams. Seems like a really trivial amount. Why is it that the body in the person without diabetes seems to be able to keep it at, you know, 80 to 100 milligrams per deciliter overnight while you're fasting, but in a disease that's going to more than double your risk of mortality and increase your risk of cancer, Alzheimer's disease, cardiovascular disease, I mean, it's really a problem for your health. It's only doubling the amount of glucose in there, and it's still a relatively trivial amount that needs a constant, constant update. How can we explain this delta that seems so trivial in the absolute amount that could be consumed in just an extra couple of minutes, but yet the steady state is still off by this factor? What's going on and why?

When you think about why is that a disease problem with only a twofold excursion, then you think that, right, the system has been built to have about the most circulating glucose that you can have safely. And I think a lot of the really important metabolites have been kind of pushed to the edge this way. And so we know that there are deleterious protein modification reactions, glycosylation reactions that occur when glucose gets above this point. So we kind of, in evolution, pushed right up to the highest, non problematic glucose. We didn't do that for a lot of other metabolites. And that's why there's not a lot of part of why there's not a lot of other diseases like diabetes. So that's part of the answer is evolution didn't build a lot of wiggle room for your glucose to safely rise, because having that good amount of glucose circulating is really valuable. Every time your heart pumps, it's sending that amount of glucose to tissues, and that's productive. On the flip side, obviously, there's a broader set of derangements in the body to produce this twofold excursion in glucose. And this has to do with things going wrong in fat and fat handling. And so that's part of this whole metabolic syndrome that leads to the full set of downstream health consequences.

Again, I think this speaks to why the flux problem is the more interesting problem than the static problem. Because if you just think about this example in the static context, you would say, okay, well, at 7:04 and 3 seconds a.m. your blood sugar is 190, but three minutes later, if nothing changes, meaning if your liver doesn't put too much glucose back into circulation, you'll be fine. So the problem is not that your blood sugar is too high in that moment. The problem is the liver assumes in part that that's the right level. And it continues to do it because at that moment when you're not eating, that is the only source by which glucose is getting into the bloodstream. So we're maintaining this elevated cycle. It's everything from gluconeogenesis, hepatic glucose output. I mean, all of these things continue to stay unregulated. And I think that's only really appreciated when you think of time and the passage of time.

I think one thing that's really interesting is that you can have, depending on the details of how processes are tuned, the same amount of production and the same amount of consumption. And these have to be balanced for your glucose to stay anywhere close to steady. So in a diabetic, production and consumption are balanced. In a healthy person, production and consumption are balanced and they can even be the same amount of production and consumption. But it can just be that you need a higher amount of glucose to achieve that same balance of production and consumption in the diabetic. And that reflects, in my opinion, at least, underlying issues with how fat is handled, that you either need more glucose to induce more insulin in order to suppress lipolysis in the diabetic, or more glucose to out compete fat to get burnt in tissues. A lot of what's setting blood glucose is competition between glucose and fat. This is a very old idea called the Randall hypothesis, but I think there's a lot of truth to it. We have a lot of new data that's consistent with it. A lot of these issues come back to making room for glucose to be burned by controlling the amount of fat that's being used by tissues.

Can you state for folks the Randall hypothesis? I'd love to actually talk a little bit about the more recent data. I mean, this is over 50 years old, isn't it?

I'm a terrible historian, so I'm going to trust you on that one. But the essence of the hypothesis is that fat is somewhere between a preferred and the preferred fuel for tissues. And there's competition between carbohydrates, classically glucose and fat for burning. And so when fat is available, then glucose tends not to be burned effectively, and that's a possible cause of diabetes.

When you say fat, you don't mean fat within adipose tissue. You mean fat that's available for use.

Fat that's available for use, and so that can come in multiple forms. The simplest way to think about it is free fatty acids that are floating in the bloodstream, and that may be the most important, important form of it. Also, adipose stores within tissues, not subcutaneous white adipose, which is typically a healthy place to store fat molecules. But you can end up with what people call ectopic fat, for example, droplets of fat building up in muscle. And when those are there, they can compete with carbohydrate for being burned, or you can have breakdown of lipoproteins from the bloodstream. Things like VLDL we desperately need to have broken down in order to have a good HDL and a low LDL cholesterol.

Tell me about some of the more recent evidence around why that hypothesis may be more compelling, even so than when it was proposed.

We've been doing experiments that look at what are things that can suppress glucose use in tissues. One thing we see is it's just very clear that fat does this. We're certainly not the only people to do this. I think there's a long history of this, but it maybe hasn't been adequately appreciated just how fundamental that result is. And if you turn off lipolysis different ways, then you rapidly induce glucose consumption, and if you provide other alternative fuels. And we've learned that lactate is a very important circulating fuel, and so it also will compete with glucose to suppress glucose use. So the fact that you can have multiple different types of fuels, either fat or lactate, and any of them will suppress glucose use really makes me believe in this kind of competitive nutrient environment and that that plays a central role in determining whether you clear or don't clear glucose and how high your glucose has to go basically in order to be cleared.

So let's talk a little bit about lactate, because this is one of those things where now, given how much I think about lactate, read about lactate, and had a number of podcasts where we get into some detail. Either I was asleep through part of medical school, or it just really wasn't presented in anything other than the following. When your demand for ATP gets high enough and quick enough, you're gonna basically take glucose and when you turn it into pyruvate, rather than take the efficient path of shuttling pyruvate into acetyl COA through the Krebs cycle, where you can generate lots of ATP requiring oxygen, you're going to take a quicker path that's less efficient, but doesn't require the same cellular oxygen. And you'll turn pyruvate into lactate, you won't get nearly as much ATP. And you'll also tend to generate a lot of lactate, which tends to gravitate with hydrogen ions, which tends to kind of poison the muscle a little bit. And that's why it becomes rate limiting in terms of how long you can sustain that level of output. Maybe explain today why that's the tip of the iceberg in a generous sense of the term.

I think it's all actually really important stuff. It's just only, as you say, part of the picture. And I think the other part of the picture is that mammals have been wired to use lactate as a major circulating nutrient. It's a super, super fast turnover nutrient. So when you think about that glucose, and you're having a few minutes supply circulating in your blood lactate, you have even shorter supply than that. It's constantly being made, released into the bloodstream and consumed. It serves as an almost universal nutrient. There are transporters that will carry it into virtually any cell in your body.

These are the mch, or is it the MCT transporters?

These are called MCT transporters, stands for monocarboxylate, because lactate has one carboxylic acid, if you think of it as a chemistry perspect. And so those transporters are ubiquitously expressed and they allow lactate basically to go everywhere.

Which, by the way, Josh, that already differs from kind of how we learned it in biochemistry class, which was all that lactate goes back to the liver and the CORI cycle turns lactate back into glucose and then just exports it down the glucose pathway via hepatic glucose output. And you're saying, I'd like to understand when that happens versus when it comes to each other. Tissue says, oh great, more fuel. Let me take in this lactate.

I think the really important thing about lactate is that glucose penetration into tissues is actually heavily regulated. It has to be heavily regulated so that if we go through A period of having low carb intake, there's still glucose preserved for the brain and for other cells that particularly need it. And lactate is the universally available form of carbohydrate in healthy heart, at least in the fasted state. It basically will not touch glucose, but it will use lactate as fuel.

So its preferred fuel is free fatty acid. Would that be.

Its preferred fuel is free fatty acids. It probably also gets some fatty acids from lipoproteins. And it definitely uses lactate and also things like ketone bodies. This is like a very clear example of a tissue other than Liverpool that net consumes lactate, just using it as a fuel to have access to carbohydrate energy.

Now, lactate is another metabolite that I pay a lot of attention to, Josh. So as regularly as I'm checking my glucose, I'm checking my lactate. And unlike glucose, the range is much greater. The lowest glucose I've ever measured in myself is probably 50 milligrams per deciliter, and the highest, not including the time Jerry Reeven had me do an insulin suppression test at Stanford and I almost died. Actually, this is actually a ridiculous story because one of the IVs got blown and we didn't know that they were pushing glucose because I was just getting so hypoglycemic. I could feel it. You know, you learn in medical school what hypoglycemia feels like and when you start sweating really profusely. And this was like nothing I've ever experienced. It felt like a bucket of water got dumped on me and I was like, they gotta push glucose. And I could feel the IV was blown. Anyway, to make a long story short, when they finally corrected it, my glucose got up to 240 milligrams per deciliter. Call it 250. That's a 5x range. But with lactate, I mean, I've measured it as low as 0.3 millimole and as high as 20 millimole. So that's a 60 fold difference.

Big range. But it probably depends a lot on your physiological state.

Well, of course the 0.3 would be at rest and fasted. The 20 is kind of an all out 2 minute effort. But the point here is that's a much bigger range. Is this regulated? Is there an upper limit to how high lactate can go? Or is it simply how much pain you can tolerate in terms of what is necessary to generate lactate? Is there truly an upper limit?

I'm honestly not sure. You may be right on the pain side of the scale. This has to do with how fast its production and consumption are. Right. So you can have that excursion to 20 that can be cleaned up in a few minutes if you're actually, you know, completely resting.

But it goes down very quickly.

Yeah, this is a very flexible metabolite. This way.

I remember first reading about this in about 2011, where people were starting to say, hey, neurons might like lactate besides glucose. Because at that point in time, there were really only two fuels that a neuron would ingest. Right. So under normal circumstances, it was exclusively glucose. And then George Cahill showed in the 60s. Yeah, but if you starve somebody, you can turn up to 60% of that fuel stock into beta hydroxybutyrate. I think it was bhb, maybe it was acetoacetate, but it was a ketone. So you'd be maybe 60, 40 in favor of a ketone to glucose. But that was really it. And then there were these kind of whisperings and these animal studies that suggested, no, actually neurons will consume lactate. Where are we today on that front?

I think it's still very unclear which cell types in the brain are the lactate consumers versus lactate producers. Certainly there's lactate use in the brain.

And is it more astrocytes neurons do we know?

I think it's really an active area investigation. I bring biases to it, but I don't bring answers. You know, my bias is that we are a neuron centric form of thinker. Right. And we didn't evolve to make glucose a unique brain fuel in order to feed astrocyte. We did it to feed neurons. I do think there's a special neuronal dependence on glucose, but lactate goes everywhere, so it probably goes into both astrocytes and neurons as a fuel in the right circumstances. And it probably can be excreted from both as a waste, depending on exactly what activities are required in the brain at that time. And that's really the beauty of lactate, is it allows you a tremendous degree of flexibility that wouldn't exist otherwise. And this was actually thought about a lot by a guy named Brooks at Berkeley, who.

George Brooks.

Yeah. Recognized ubiquitous potential for lactate as a fuel. We were able to contribute to that story by really showing it using mass spectrometry to make it crystal clear that this usage happens throughout the whole body.

What is the evolutionary reason in your mind for why the body would allow most tissues to love lactate as a fuel directly versus just having the liver mop it up at the same Kinetic rate, turn it into glucose and shoot that glucose out. Is there an obvious reason for why the current strategy is a better one?

It's not an easy answer, but I think there are strong reasons. And I'll say that we've lately done experiments in yeast. Actually, yeast make ethanol waste, and people always, I think, assume that yeast face this exact same choice that you talked about. When you get to the level of pyruvate, either do you spit it out as a redox balanced waste. In humans that's done as lactate, in yeast that's done as ethanol, or do you take the pyruvate into the TCA cycle? We see that going all the way back to yeast, that's a false choice. The default is to spit out the redox balanced waste, and then you can always pick the waste up and reuse it if you need energy from the TCA cycle. And so I think this goes back to the very earliest days of eukaryotic life. Basically, that you want to be able to run glycolysis whenever you need to run glycolysis. So use glucose. Whenever you need to use glucose that takes you to pyruvate, you've created a redox problem because you have electrons from the glucose that are not sitting on the pyruvate. The first priority is always to solve that redox problem that's achieved in our bodies by spitting out lactate. You don't really want to hold that problem within cells in your body. You want to get that all the way onto the circulation. So every cell in your body can work on this master metabolic challenge of keeping electrons balanced. Then whoever needs energy, and these electrons are a super valuable form of, of energy, can pick them up in the form of lactate. There's been kind of this false coupling of oxidative and glycolytic metabolism in the way biochemistry is taught, when really our bodies, eukaryotes all the way back to yeast, are really designed to be much more flexible to allow these two processes to happen in yeast completely independently, because they really can just spit out ethanol to the environment in us, quasi independently. So independently at the level of individual cells in our body. So none of them faces this pressure. And that's really good. So if you have a bout of hypoxia, okay, you can release lactate and elsewhere in the body, the problem can be cleaned up. Now in our bodies, it has to be cleaned up within the body somewhere, because we don't have any master release valve for this. So all of our cells together have.

To Solve this problem, meaning the way that yeast can literally eject ethanol from their cell and get it away. We can't emit lactate from our body, we can emit it from a cell, but it's still part of the broader system.

Exactly. And that's when you get into medical problems like lactic acidosis. If you have a very fundamental metabolic deficiency or if God forbid, someone put a bag over your head and you couldn't breathe, then you end up in this crisis of redox imbalance. But we distribute that problem across the body through this concentrating lactate in and out of cells and letting whatever cells that need carbohydrate energy use the lactate. The system would be way, way less flexible. If only the liver could clean this up. Would be also way less commensurate with effective burst exercise. The heart is super well perfused. If less well perfused muscle, that's far from the heart. Okay, so it's hard to get oxygen there is making a lot of lactate. Of course, it's very advantageous at that moment for the heart, which is sitting on more oxygen than it needs to use lactate rather than fatty acids, which are better long term things to store for the future anyway. So it's way, way better to have the system designed this way.

And is that regulated then locally? Is that regulated at each cell? Like how is that decision made? Because how does that myocyte in the heart know that the energy of the entire system so that it can make the decision that in the short run.

Counterintuitive seems medically, or maybe textbook med school counterintuitive. But it's physical chemistry, pure intuition. Lactate goes up, it gets burnt.

But how is the decision made? Are you saying it's just made on mass balance and availability of substrate?

It's made on mass action and availability of substrates. So there's no decision.

It's basically a gradient problem across the board.

If you have too much lactate, it flows out. If you are short on energy, it flows in.

One thing that I've become very interested in clinically is the implication of fasting lactate levels in the population. So if you measure a person's lactate level first thing in the morning, you're going to see quite a bit of variability and it seems to be proportional to their metabolic health. The higher that number, the less metabolically healthy they are. It's not uncommon in someone who's insulin resistant to see fasting lactate levels approaching 2 millimole with no activity. Whereas in a healthy individual, it'll be below 0.5 millimole. What do you think that tells us about fuel partitioning and this problem of metabolomics?

I think there's a correlation between fasting glucose and fasting lactate. But lactate is maybe harder to measure, but perhaps even more intimately tied to the essence of metabolic dysfunction molecule. It reads out a few things when lactate is high. It reflects the fact that during these times of fasting, when glucose is not really supposed to being used much, you're still using too much glucose, converting too much of it to lactate, and at the same time that your lactate clearance system isn't working very well. And typically that's because you're having competition between lactate and fat to be burned. This all feeds into the syndrome of diabetes.

Now, there's another interesting push observation that I've made, which is, I wake up in the morning, check a lactate, it's 0.4 millimole. I eat the biggest carbohydrate meal I can ingest, don't lift a finger other than to feed myself. I recheck my lactate in an hour, it's one millimole. Why did that happen? We can understand the biochemistry, which is, I have more glucose to metabolize. But this gets back to your point of med school. Biochemistry would suggest my lactate should not have gone up. I'm taking glucose, I'm making pyruvate, I have endless cellular oxygen. I should be running that pyruvate through the Krebs cycle, and I shouldn't see any uptick in lact. But that's exactly what I don't see.

Circulating lactate is an intermediary in glucose catabolism. That's just the way the body works. It's not what we were taught in med school.

Do you have a sense of how it's being taught today? Do you get the sense that biochemistry students at Princeton and Stanford today are being taught what we were taught with respect to this sort of more rigid model of lactate and as a metabolite?

Well, I'm probably chipping away at it at Princeton, but I don't know how much it's shifting at the medical education level yet.

Probably.

Thank God I haven't sat through those classes at Stanford. Again, I think it's something that we should see shift, and I hope we see the next generation of biochemistry textbooks talk about circulating lactate as an intermediary in glucose catabolism. I think that's a really fundamental thing for people who want to just think about metabolism accurately to know.

And I think it's a very interesting thing to consider from a prognostic standpoint. When you go back and look at Jerry Reaven's five criteria for what was then Syndrome X and what is now metabolic syndrome, fasting glucose is still one of them. You could make a case that fasting lactate would be more telling.

I think the challenge with lactate is that it is a metabolite that can get up and down faster. And one response to stress is to rapidly convert glucose into lactate. That's just part of your body activating. But of course, there are people who have stress at a blood draw. And because lactate is a little bit more fluctuating this way, there are going to be pros and cons medically, in terms of using it as a biomarker. I don't think our problem with metabolic syndrome anyway is diagnosing it. Our problem is preventing it.

Yeah. Although what I would argue is I think we treat metabolic syndrome too discreetly and I think we come to it too late. I think we should be looking for things far before you actually have hypertension and truncal obesity and dyslipidemia and hyperglycemia. And I do wonder with nothing other than just intuition if lactate dysregulation, for lack of a better word, might be one of the earlier canaries in the coal mine.

I totally agree with that.

I want to go back to something that we've talked about a couple of times. You've mentioned it in passing. You and I know what it's about, but I think it's such an important part of where we're going to go in a discussion that I almost need you to go into full Prof. Mode and really explain two things which are obviously highly related. In a moment, you'll see the first is how the electron transport chain works. What is the Krebs cycle doing, and how is that feeding into this massive generation of energy currency? And specifically, can you talk about it with special attention to the concept of redox? I would encourage you, Josh, to take as much time as you need, because the more the listeners understand this, the more they'll be able to understand nad, nadp, nadph, nr, nmn, all of these other things that people really care about. But I think, unfortunately, they've been conditioned into very glib understandings of these things, which I think are serving no one any benefit, without actually going back to understanding the root of this problem.

Think of it this way. Fundamentally, you eat three Macronutrients, carbs and protein and fat. And in a healthy adult first approximation, every carbon atom that you eat in any of those three forms needs to exit your body as exhaled carbon dioxide. And all of that exhaled carbon dioxide, first approximation is made in the TCA cycle. The main way that nutrients flow into the TCA cycle to become carbon dioxide is first turning into pieces that are two carbon units in size. And so from carbohydrate, the basic flow is glucose to lactate and then lactate to pyruvate to a two carbon piece that goes into the TCA cycle. Fat is basically composed of pre assembled two carbon pieces. So they just get chopped up two carbon pieces at a time. And the protein part is a little more complicated. We can probably skip it.

Worth just sort of noting, Josh, that protein, really, the primary role of protein is actually the nitrogen side, which we're putting into these amino acids that are building blocks. It's really less of an energy substrate, but it does have that carboxylic acid on it that still has to go through this cycle and be exhaled. In other words, that's why we'll skip it for now, because it's really not a significant energetic component. Right?

I think it really depends on the kind of diet you eat.

True. If you're on a carnivore diet, then it's probably a different situation.

It's a very interesting side discussion. But ultimately, unless you're gaining protein mass, which of course wonderful for us guys anyway, when that happens, typically at least societally, smiles on it. But other than that, you know, whatever amino acid carbon you take in the form of protein has to be balanced with also amino acid catabolism. At that level, it's not that different than carbs and fat. It's just a little different in that it can enter the TCA cycle sometimes also as four carbon pieces. But a lot of amino acids are broken down into these same two carbon pieces. There are just 20 of them. So no one wants to hear a discussion of how all 20 of them get chopped up. So ultimately you end up with these two carbon pieces. They congeal with a four carbon piece and that makes citrate. One of the problems with the Krebs cycle is that it has three names. The Krebs cycle, in honor of the amazing biochemist who played a key role in figuring it out. The citric acid cycle. And that's named for this condensation molecule of the four carbon and the two carbon piece citrate, or the TCA cycle and TCA is tricarboxylic acid, and that's because citrate has three carboxylic acids. Okay, so you have this cycle that unfortunately has three names, but it's probably three times as important as anything else in metabolism, so maybe it's fair. Ultimately, as this cycle turns, it's going to spit off the two carbon pieces that came in as carbon dioxide. And in so doing, it's going to take the electrons that were part of those two carbon pieces and pass them to this famous cofactor, nad, to make nadh. That H stands for hydrogen, and that hydrogen is really one proton and two electrons. And so this is another confusing nomenclature thing that you just can think of, that H, even though it may sound to those who've taken freshman chemistry, like H plus like acid, this is an H with two electrons stuck to it. So it's really what we call hydride, or electrical form of chemical energy. Then nadh that's made from NAD there is what feeds into the electron transport chain. And those electrons then flow through a series of proteins that sit in the inner mitochondrial membrane. The mitochondria have two membranes. The outer one is kind of leaky and kind of not so important. The inner one is super tight and has a ton of regulation in it, and most importantly, can be used to pump protons to one side or the other. And ultimately, it's the pumping of protons out of the mitochondria that's the function of the electron transport chain. And in this kind of metabolic flux way that we talked about earlier, the protons that get pumped out just flow right back in. But as they flow back in, they turn a turnstile. And as that turnstile turns, it squeezes adp, an inorganic phosphate, physically squeezes them together to make ATP, the master energy currency that we use to power our neurons for thinking, our muscles for moving, and so on.

One of the things about this system that is just so beautiful is the transition from chemical energy to electrical energy, back to chemical energy. You know, I've tried to explain this to my daughter. She's 13. She's not fully in love with it yet, but I know at some point it'll be a more fun discussion. But it really is a miracle, right? So much of biology just seems like it's hard to believe it all worked out. But if you were going to rank all the things that I can't believe it worked out, this has got to be in the top five. Like, let's go back to the basics again. You eat a piece of Bread, you're eating glucose. It has these carbon to carbon bonds and carbon to hydrogen bonds and some carbon to oxygen bonds. Now refresh my memory. But carbon to oxygen is not a very energetic bond. Right?

Co double bonds are spectacular bonds. They're super high energy. But that's where life, I shouldn't say life, that's where physics and chemistry want to flow too. They want to make these high energy bonds. And in making high energy bonds, you can release a lot of energy energy. Those are bonds that are very energetically favorable. So they're the end state. It's the C H bonds, as you're alluding to that start out energetically loaded, okay? They're less energetically good in and of themselves. So they have the potential to become something better.

I'm glad you're adding this level of chemical rigor to this. The point I want to make is these carbon, carbon, carbon hydrogen bonds have this potential that this entire cycle with three names that's so wonderful, basically liberates. It basically says we're going to take that chemical energy and we're going to liberate it through electron transferring apparati. And then at the last second, we're basically going to quickly shunt it right back into a chemical bond, which is the P binding to the ADP to make the ATP. And now we have this energy currency that is going to go and do its own thing. And it has lots of different ways that it unleashes itself. So explain to people the difference between oxidation and reduction in chemical terms, because I think people have to at least hear once, what's an oxidation reaction? What's a reduction reaction? And then why we use the term redox synonymously with these two oxidation reduction.

Are always coupled, okay? And they refer to movement of electrons. And so when electrons go from substance A to substance B, the one that gives up the electrons is oxidized. It's subject to oxidation. The one that receives the electrons is reduced. It's the subject of reduction.

Let's talk about redox pairing. So you've already brought up NAD and nadh. So talk about how oxidation, reduction pairing works with those two to facilitate the electron transfer down this lovely chain of the inner mitochondrial membrane.

This is a pair where NAD is the oxidized form. NADH is the electron holding or reduced form. It normally exists in a quite biased ratio towards a lot of NAD and a small amount of nadh. The way nature works is that whenever any pair of chemicals is skewed in one direction, it's favorable to turn the one that's abundant into the one that's less abundant. And so this makes NAD a decent electron acceptor. And so it's sitting there prepared to pick up electrons from these intermediates, carbon intermediates of the TCA cycle, that are coming from carbohydrate and fat, and take the electrons, make nadh, which then can feed into electron transport chain. And that backend has to happen fast in order to keep this ratio skewed. So you have mainly NAD and not too much nadh. And that's really important because when that NADH starts creeping up, all sorts of things start going wrong, such as NADH going up will drive too many electrons into the electron transport chain. And going back to nav, you know, he's done a spectacular job showing how that leads to production of free radicals. You need the right amount of this. But this is a clear way to get toxic amounts of free radicals if you have NADH buildup. Secondly, it just gums up metabolism. If you have too much NadH relative to NAD, you can get into problems not having enough ATP. And so it can also make signaling things go awry.

Now, are there clinical scenarios in which we see that happen, or are these more typically, they're things that result from toxicities. The classic thing you learn in medical school to explain the significance of this whole system is cyanide. Maybe tell folks how cyanide works. And I don't know if that's too extreme an example of how this system can be hijacked, but let's see.

Cyanide is an electron transport chain inhibitor. And so that leads to the whole system just backing up bit by bit. And so you can't then transfer electrons from NADH into the electron transport chain. And so NADH goes way up, NAD falls to the floor, and then you have no way to make ATP. And that unfortunately leads to rapid mortality.

That's an interesting point, because this again comes back to the kinetics and the flux, which is it's not like cyanide kills you in an hour. I mean, it kills you in seconds. It's really a sobering thought.

ATP turnover via this system is on the timescale of a second. So same for nadh. And so these are things that are just whizzing through our bodies all the time and that we're constantly dependent on.

Are there less extreme examples, Josh, of things that will put that balance in the wrong direction kind of chronically?

I don't want to give the misimpression that the right thing is to have as Much NAD and as little NADH as possible. First of all, it's designed to be a dynamic system. If you undergo intense exercise, you're going to drive NADH up. And this is a very healthy context for doing this transiently. Metformin, of course, is a super interesting medication and probably works mainly by slowing the conversion of NadH back to NAD, by impairing the complex one of the electron transport chain, the one that does this initial electron offloading from NADH to make nad.

How well is that understood? I mean, you'll talk to five people who study metformin and they'll tell you five different things, which I think just tells you how much we don't know. But I don't think it's really disputed that metformin inhibits complex one, is it? I think the broader question is how much is that the main attraction versus kind of a sideshow.

There are many people who know more about this than me, but one thing that we tend to do in our lab sometimes is take these famous metabolic effects like metformin inhibiting complex 1 and just do a quick test of it. And I say when we do that, about half the time they look to be true and half the time they look to be dubious. And metformin was a shining star in our hands in inhibiting complex I. It was one of the cases where I really felt like it may do other things, but it certainly does what it's supposed to do there. It does that strongly. And I think it's probably the fact that it does it in a relatively liver specific way due to the way that metformin enters cells of the body that leads to it, first of all being safe. There are many things that make it safer than cyanide, but it is really crazy that maybe the world's most widely used medication is at some level inhibits the electron trans. It's a mechanistic analog of cyanide. So you have like one of the most acutely lethal substances and most widely used drug working in a remarkably similar way. And so I think the fact that there's a strong liver specificity is probably what makes it net beneficial for at least a subset of people.

What's the change that you saw in your lab, Josh? So if you go off metformin, what's your NADH to NAD ratio? And then on metformin, how much did it change?

That depends how much metformin you use.

But if you tried to approximate an actual clinical dose of say a couple.

Grams a day, I'm not sure we did this in a way that I would consider clinically applicable. It's certainly crystal clear that it goes in the right direction.

And so it doesn't surprise you that metformin would raise fasting lactate levels, correct?

No. I mean, it certainly is aligned to.

Do that and that's just backing up further. It's just basically creating more of a roadblock into the tca is going to give you more lactate.

Yep, more of a roadblock and electron disposal, basically.

Do you think that that's a neutral effect or do you think that that's a potentially deleterious effect of metformin that is probably offset in a patient with diabetes by the benefits that it has on hepatic glucose output?

It's a great question. I don't think. I know. I think having more circulating lactate can be a bit of a challenge for clearing fat because they have some sort of competition. So from that perspective, I think being in a lower state might have some benefits. But then, you know, lactate is a valuable fuel as long as it's not getting too high levels. I'm not sure how that all plays out in terms of long term health.

Anything you want to say about NADP and nadph? Just to round it out so people know the full story.

These are super important cofactors. They live just on the edge of what people who take biochemistry, either in undergraduate or med school, learn about or don't learn about. They're fascinating cofactors because in terms of their intrinsic chemistry, all their intrinsic chemistry from the energy point of view, is exactly the same as nad, nadh, but they have a different handle on them chemically that allows biology to use them in a different way. The ratio is maintained, quite different level from nad, nadh. So NAD nadh is super biased towards nad. This is much more of an even pairing, which means there's much more driving force to dump the electrons off rather than to absorb them up. NADPH is really, to me, second only to ATP, a master energetic building material. And it's the building material that's used, for example, to assemble fat. It's the most important one. So as you take pieces, two carbon pieces from carbohydrate and want to put them together to make fat, you keep dumping in electrical energy in the form of nadph. And then NADPH is used in all sorts of other really interesting ways to fight reactive oxygen species. It's also used if you're trying to kill bacteria, to intentionally make reactive oxygen species. This is where biology is freaking confusing and complicated and there's definitely the yin and yang that you have this awesome cofactor that's so important for fighting oxidative stress and also can be used to create boatloads oxidative stress intentionally when it's needed.

First of all, that was a fantastic overview of how the Krebs cycle works, and specifically with attention to how electrons move through it and move through these redox factors. Which then brings us to a part of the discussion where a lot of people have an enormous interest, which is, I don't know, go back seven, eight years, it started to become fashionable, and it's only become more fashionable to talk about supplementing with nad. I say that quote, unquote, and we're going to talk about why you don't actually supplement with nad. But is it safe to say that at least part of the impetus for this was the observation that as we age, cellular NAD levels decline? And you've already made a very compelling case for why NAD is important. I almost want to avoid the whole sirtuin side of this because I think that story keeps changing. So unless you feel strongly or compelled to get into sirtuins, we can put those aside for the moment.

Yeah, I love putting sirtuins aside. The first principles in this field are great. NAD plays this super central role in energy generation that we all want to feel more energetic, whether you want to be a more extremely successful athlete at age 21 or whether you want to feel at age 50 like I am, or later like you're 21. So you think we could just turn up the burner capacity. Right. This would be absolutely fantastic. And then we have this data that NAD is depleted with aging. Although I have to say, when we do those measurements, we agree that NAD is depleted with aging. But it is a lot more subtle than you would think. Looking at the literature. These are really quite subtle NAD depletions. You know, we were talking about these ranges earlier, the kind of threefold range where a lot of metabolites live on a daily basis, some of them glucose. That's their worst day. Right. And as you pointed out, some of them, like lactate, they may do the threefold all the time, and then the 60 fold when you stress them. NAD changes we see with aging are like 10%, 20%.

Oh, wow. I didn't realize it was that little Josh.

And I'm not saying that in some tissue of an aged human, there might not be bigger effects. This is the first caution I would give to people thinking that they're going to fix everything through nad. On one hand, it's a robust finding that this is something that changes with aging, with a central metabolic role. On the other hand, it's something that happens with a fair amount of subtlety.

Can you explain to folks how this is done? Because we talk about measurement sometimes a little too glibly, it's pretty easy to explain how we can measure glucose and hemoglobin and lactate. At the other end of the spectrum, we've talked a lot about ATP. What I think most people don't understand is it's very difficult to measure ATP. It comes back to what you said a moment ago. This ain't sticking around a very long time. You're using Mrs. And super complicated physics to be able to measure these things. Where does NAD fit on that spectrum and how do you actually measure it?

Good news is that nad, unlike nadh, is not like one of these super transient metabolites. And so NADH measurement is really wickedly difficult. But most of this NADH NAD pair sits as nad, and it tends to sit around for hourish timescale.

Oh, so you don't necessarily have to flash freeze tissue or things like that?

That's right. You have some more flexibility in making those measurements as long as you're not irritating the tissue in a way that leads to massive NAD degradation, which people may do sometimes by accident, but I think generally it's not that hard of a measurement. Nad, obviously, like ATP, it's a tissue metabolite, not a circulating metabolite. So you need biopsy specimens to measure it. I'm not a master of the literature of NAD levels in human tissues, but my not fully informed perspective is that it probably isn't as much as we should have. And that's because it's hard to get biopsies from people.

If you take blood, if you take whole blood and you look at pbmc, can you look at NAD levels in there with relative ease, or is it too complicated because by the time you separate the pbmc, you've kind of lost your window?

I think it's a really good question because there is quite active NAD metabolism in immune cells. I'm not an expert in this. I bet there's a way you could develop a good protocol. I haven't followed how good the measurements up to now have been.

So most of what you've measured has been in tissue.

Typically, you know, we work a lot in mouse, sometimes we measure human, but more typically on the cancer side there, we just take tissues and freeze them and extract metabolites, do mass spec and.

So you're seeing a consistent, clear decline in NAD with the aging animal or human. But it's not a fold reduction. It's a percent reduction, 10%, 20% reduction.

That's the most common thing. Yeah.

This generates a hypothesis. The hypothesis is if you restore NAD levels in the old organism to the level that they were in the young organism, the old organism will feel and perform like the young organism. That's one hypothesis. Another hypothesis would be if you induce supranormal levels of NAD in any organism, they will feel supranormal. Let's assume that both of those hypotheses are simultaneously testable. What happened? Five, seven, eight years ago, NAD clinics started popping up all over the place, and they started saying, if you come here, we'll put IVs in you and we'll give you NAD. So let's first explain. Why did they do this intravenously? Why couldn't they make an NAD pill?

NAD and its precursors are broken down in the gastrointestinal tract. And so if you take nr, for example, so nicotinamide riboside orally, it mainly will enter the body in the form of nicotinic acid or niacin, which is a healthy substance, nothing wrong with it. But except for maybe the epithelium or gastrointestinal tract, the body is not seeing nicotinamide riboside, and nad.

Just to be clear, we're going to talk about NR and NMN in a moment. But nad, there's no way to orally take it.

There's no known absorption route for nad, and I think it'll get broken down probably all the way to nicotine, nicotinic acid, although I'm not 100% sure anyone has proven that. I certainly don't think it would enter the body any other way than either nicotinamide, which is like a little bit closer to remaking nad, or nicotinic acid.

So what happens when a person receives intravenous nad? What's the fate of that nad? One of the things in metabolism and biology is anytime you put something in a vein, you bypass the liver with something called the first pass effect, which in your former life was very important, because when you had these patients in the ER that you were giving inhaled drugs to, it's not just the speed with which they were getting it, it's that you could actually deliver the exact drug you wanted, not a pro drug that could be modified by the liver. So this idea of giving intravenous NAD is at least theoretically interesting because you're putting the molecule of interest directly into the venous system. So what's its fate?

You may be more up on this than me, but it's going to get broken down partially because there's not clear uptake mechanisms known anyway to get NAD from the bloodstream into cells. Nicotinamide mononucleotide may be able to enter cells directly, or nicotinamide ribosides. These are partially broken down forms of nad, but that are nevertheless meaningfully closer to NAD than the normal things that circulate in good amounts in our bloodstream. That does partially, I would say, short circuit the route to cells making nad, so they kind of can break down partially the NAD in the bloodstream, take these partially broken down NAD precursors into cells and rebuild NAD in a shortcut manner that probably has a good chance to bolster NAD levels.

So in other words, when you give intravenous nad, there is no transporter to take NAD into a cell. But that NAD breaks down into things like NR and nmn. And in the vascular system, we know that those things can get taken up at least into some cells. Do we know which cells have the capacity to do that or which cells don't?

Certainly at least some important cell types in the body can take those up, maybe pretty broadly, but I don't know off the top of my head.

And so then when the NR or NMN gets into this cell, is it relatively straightforward that it will be reconstituted into nad? What's the energetic cost of doing that, or how easy is that? And is that the favored reaction at that point?

Yeah, I think it's the favored reaction, which is the important thing. And this is not a big demand, relatively speaking. The big energy flow is through this NAD and NADH exchange. But the making of NAD itself is not an expensive process per se. NAD stands for nicotinamide adenine dinucleotide. It's two kind of nucleotide pieces put together. And when you take in NR or nmn, it's one of those two pieces. But the more interesting side, and the other side comes from ATP, and it's there all the time because all your cells have ATP. Or you got much, much deeper problems. And so you just snap it together. And I think you end up with probably effective NAD supplementation when you go the IV route.

In other words, taking IV NAD will probably increase intracellular NAD levels, though not directly because there's not a transporter. But it goes through this sort of circuitous route to get there. So might not be the most efficient way to do it. But this Certainly corrects a statement I've made in the past, which is intravenous NAD is not a good way to get NAD because we don't have a transporter. That's correct, but incomplete.

That's a fair summary from my perspective.

In other words, we don't really know how much. If you take 100 units of NAD intravenously infused, we don't really know how many units ultimately make their way into a state cell, but it's probably not 100.

I'm sure there's a fair amount of loss in the process. There's a very interesting protein called CD38 that's I think, designed to control these kind of pathways. It's a suppressor of NAD levels. It works, I think, by breaking down NAD that's outside of cells. And mainly there's not normally in physiology NAD in meaningful amounts, probably outside of cells, but there is NMN in meaningful amounts. This is a protein that's super good at breaking down nmn. It still leaves you with nr, so it's one step further away from being nad, but it's still meaningfully closer than your typical physiological precursor. And so I think it's positioned to, as you say, at least some places in the body, boost nad.

Today, I think the majority of efforts to increase intracellular NAD are done through oral precursors. And the two are NR and nmn, which as you said, are pretty similar. And are you aware of a more convincing argument for why one might be a more preferred substrate? I haven't particularly seen arguments that one is superior than the other. I've seen some unpublished data that suggests one can be made more temperature and moisture stable than the other. But let's put that aside for the moment. Would you consider these two, a first approximation, equivalent approaches?

Yeah.

Okay, so now talk about something else that you said that is also kind of news to me, which is what is the effect of the gastrointestinal tract on these agents?

I mean, they get broken down and they get broken down all the way to the level of nicotinic acid or niacin. Basically this is the main way they enter the body. It doesn't mean that there can't be a trickle of them entering some other way that has a physiological effect or that there's some local effect or some effect on the microbiome of taking them. Biology is super complicated. There are ways that these could be doing interesting health supporting things, but I don't really think they're fundamentally different than taking a physician prescribed niacin pill. From the perspective of of providing NAD precursors.

Now a physician prescribed niacin pill. When people used to take niacin for hyperbata lipoproteinemia, it wasn't uncommon to get a real flush from the medication. Now, I don't remember what doses people were taking, but I feel like it was on the order of grams, not milligrams. Do you recall how much niacin you would need to give somebody for them to experience an actual flush?

I think it was a few grams. That's where my recollection is too.

Is that the reason people don't experience a flush with NR and NMN, because they're typically taking 500 milligrams to 1 gram and that's simply not going to produce enough niacin to reach that threshold?

Yeah, they're also kind of niacin pro drugs, so they probably are delayed absorption forms of niacin. So that may smooth things out enough so they may be better tolerated. But I think this is how I would fundamentally think about them, is that they're niacin prodrugs.

Your lab has done some of the flux work on this. What are some of the most interesting things you've learned about how NR and how nmn, when given orally end up in different tissues and what the effect is in the liver versus the muscle versus the plasma?

I think the main thing you see is that these are converted to niacin. They will raise niacin, particularly niacin, heading to the liver out of the gastrointestinal tract. So in the we call the portal circulation that connects your intestine to the liver very effectively. And that other than that, their effect on like boasting their own circulating levels is somewhere between subtle and vanishing. I'm still not sure which of those two it is. They certainly remain in the bloodstream much less abundant than nicotinamide, which is the thing the liver is normally producing to feed NAD precursor to the tissues of the body. From what we have seen, no clear route for oral NR or oral NMN to produce circulating levels of NR or NMN that are high enough to compete at least at a standard concentration level with nicotinamide, the physiological precursor as to opposed a way of feeding NAD precursors to tissues. So basically, at some level, they don't change what's happening, what most of your tissues are seeing that much, if our measurements are correct.

Where do you think you could be fooled on this? I mean, I know that's a question every scientist or every good scientist asks themselves that question. Right. How can we be fooled by our measurements. I'm sure you've thought about this. Where do you see the opportunities in this particular case to be misled?

It could be that there are local effects of NR or NMN on the intestine that are really important. It could be that their availability impacts the microbiome in important ways. The microbiome can have big effects on health. It could be that even though the amounts of NR that may reach the liver or even lower amounts that may reach the heart or something are really small, that there's a subset of cells there that are really NR preferring because maybe they're really deficient in using nicotinamide and maybe getting even small amounts of NR to those cells is meaningful. I think these are all possibilities that we're very much open to. My base assumption is that often the obvious is true, and here the obvious would be the physiological system. Just isn't that interest impacted by this particular type of oral supplement.

Do you think there's any chance that with chronic administration you'd see something different? Because I'm assuming in these experiments you're not seeing the effect of these chemicals being ingested chronically, or are you?

Human can be different than mouse. Okay, first of all, it's another important thing that I say. We haven't done these experiments in human. Someone, if they aren't already, should do these experiments in human. Yeah, chronic versus acute. So there's a bunch of variables that.

Could alter things based on what you know. Now, if the hypothesis is true, if restoring intracellular NAD levels at 50 to the level they were at when you were 20 would improve some measure of performance based on what you know today, what do you hypothesize would be the most efficient way to restore NAD levels?

IV is the promising way to do the restoration. I'm not very convinced about the first hypothesis. I think the big history of medicine you and I can debate it is that things are way more complicated than people can envision. Hormone replacement therapy is like one of the great examples. Right. It didn't turn out to.

Although it's being overturned. I think if you go back and look at the Women's Health Initiative, I think it got it wrong. It was the randomized experiment, but it was really misinterpreted.

Say a bit more about that.

Maybe I picked up on the wrong thread of where you were going, but I assumed what you were going to say was, look, the epidemiology in the 80s and 90s was that giving women hormones post menopause was a good thing. And Then the Women's Health Initiative came along and said, no, it's a bad thing. I assume that's what you were going to say. Yeah, and what I was going to say was, actually, no, I think that's actually misleading. I think if you actually go back and look at the Women's Health Initiative, it was just an awful example of how to misinterpret a study. I think there was no increase in the risk of breast cancer. And if there was any increase in the risk of breast cancer, it probably had nothing to do with the estrogen that the women were given. When you actually look, for example, at the relative risk and absolute risk different in those cohorts. So remember, the Women's Health Initiative had three. Well, technically it was two parallels, right? So you had the placebo versus estrogen only in women who did not have a uterus, and then you had placebo versus estrogen plus mpa, the synthetic progesterone. So in the estrogen only versus the placebo, there was a non statistical significant reduction in the risk of breast cancer. So there was a hazard ratio of about 0.8 or 0.79 or 0.81, something like that, but it didn't quite reach statistical significance. But trending towards estrogen actually reduced the risk of breast cancer. In the estrogen plus MPA group, there was a barely statistical significant increase in the risk of breast cancer. I think the hazard ratio was, I want to say it was about 1.24, 1.25 and the P value was exactly 0.05 or 0.049 or something like that. So at the surface you'd say, gosh, this is increasing the risk of breast cancer. And what was talked about was a 25% increase in the risk of breast cancer. To talk about the relative risk increase without talking about the absolute risk is obviously irresponsible. If you look at the absolute risk change, it was 0.1%, it was one in a thousand. And that says nothing about a lot of other methodologic issues with the study, including the fact that, in my opinion, a more plausible hypothesis was that the MPA was more the issue than the estrogen. But the estrogen gets all the attention, right? So estrogen causes breast cancer, gets the attention. If you look at subsequent studies, I don't think we see that to be the case. So I'm going to hypothesize or predict that in 10 years we'll look back at what happened to a generation of women, which I think is really unfair, you know, Basically an entire generation of women got deprived of hormones because of, I think, a really poorly interpreted study. But your point notwithstanding, sometimes the obvious is not obvious. Sorry for the digression.

No, I mean, that was super interesting. I thought there was some cardiovascular risk data in that study that was surprising, but you know it much better than me.

Yeah, I think on the cardiovascular front there probably is a slight increase in risk with oral estrogen because of the hypercoagulability. I also think it speaks to understanding the use case today. Very few women on hormone replacement therapy are given oral estrogen. The preferred route of administration is a patch, you know, something like a Vivelle dot where you're given topical estradiol and you get all of the benefits of reduction of vasomotor symptoms. The incredible benefits that you see on bone health without any of the hypercoagulability and cardiovascular. So now we actually see the reverse. Now there's a very clear trend. Not just trend, it's statistically significant. There's a very clear reduction in cardiovascular mortality.

So that's a great example where you had to give by the right route of administration in order to get the net positive health benefit. Nsaids, Advil, another one. Right. People knew they had a lot of side effects, but everybody assumed that they were kind of counterbalanced by the fact they were reducing coagulability and that this was going to be cardio protective. I don't know if you're going to tell me that you still believe that, but most people don't.

So really where you were going, I think is you were saying, look, you might even just reject the outright hypothesis, like this idea that, yeah, we do observe a 10 to 20% reduction in NAD levels as we age. You haven't even bought the first hypothesis, which is, even if I could magically deliver 20% more NAD to a 50 year old, you're not sounding very convinced that that's going to improve quality or length of life.

Not at this point in time. These things involve such complicated interplay of different organ systems and it may turn out that NAD supplementation is super valuable medically. I am completely open to that and I would love that to be the case. But I think if so, it's going to be because there are select cell types that are genuinely severely NAD depleted and that we will need to figure out how to restore NAD in those cell types and then we may see big health benefits. So I think that would be fantastic. And it's completely possible. And it's possible that the general intravenous Supplementation is hitting those cells and doing that. But I think it's equally possible that it's having some adverse effect that's going to be net negative for people. And we don't know the science well enough. And we certainly haven't done the clinical experiment well enough to give good health guidance yet.

So, two things. First, a statement. This is really interesting for me because I really stand corrected, and I just want to apologize to all the people over the years that I've said intravenous NAD is not getting in your cell. I stand corrected it indirectly, based on everything you're saying, may actually be getting into at least some cells. The second is a question which is how would you even begin to tackle that question, which is, are there certain cell subtypes that may indeed benefit from NAD boosting? I haven't really seen a single convincing clinical study in humans using either NR or nmn. That has made me excited about this. And I'm not a stranger to putting things in my body without absolute perfect information. I mean, I take rapamycin. I've been taking rapamycin for four or five years. I will be the first to admit I think we have very good evidence for that. It's not perfect. It's far from perfect. We're never going to have a definitive human clinical trial. But if I'm willing to take rapamycin, why am I not taking NR and why am I not taking nmn? And the reason is I just can't find a shred of compelling evidence to tell me to do so. And I'm in the same boat you're in. I'd love it if there was, because it's a pretty easy, safe thing to take. So what study needs to be done to help someone like me, a reluctant NAD er, get on the NAD train?

I think we need to first of all MAP better the basic pharmacology of NAD in animals and human. That's quite doable. And this is something that we as a field are doing, and there are a lot of great people doing this, but we can do more and better. We need to have the technologies to look at this at cellular resolution rather than bulk resolution. This is something we're pushing very hard to develop. The ability to take a slice of tissue and say, what's the heterogeneity of cross cells in NAD levels? I think that'll be very helpful because if we see that that's really scattershot and aging and homogeneous and young, then you have your answer that all that we need is for 1 in 10 cells at any time to be really NAD depleted. And we view that 10% reduction not as some tiny wiggle down, but as 1 in 10 cells being on the road to a catastrophic. I think this is going to be a really important measurement. I think the field will get there over the next few years, not instantly. And then ultimately we need successful clinical experiments there. There have been some really persuasive experiments in animals. For example. I think there are experiments on reversing bad outcomes after renal ischemia. And so it'd be good if we could find niche experiments where there's a very strong effect in animals. A very quick clinical readout, ischemic renal event, and you do the supplementation and you get a benefit or you don't.

How was it administered in that experiment? I don't think I remember that one.

I may not get the details of that right. I think I would just say conceptually we need to find the strongest animal proof of concept that can be translated into a small but definitive clinical trial and prove that this really can do something beneficial in the right context. And then from there you can think about kind of expanding the indication to general health betterment.

Who's the natural owner funding wise of this? Is this a question NIH is interested in? I mean indirectly through the itp? Rich Miller, Randy Strong et al have already done an NR test in their very rigorous, tried and true model. As you know, that failed, so NR did not extend life. Is NIH still interested in this question enough to continue funding it? Where is your funding for this level of investigation coming from?

We mainly try to help the real NAD expert labs by doing the flux studies, facilitating measurements, but it's not the bread and butter of my life. I'm probably about an observer at the same level as you of this field. Broadly speaking, there's money at NIH for interesting science. And so this is too central to metabolism and too much public health interest for the waters to run dry.

You're not worried this is not going to run up against the funding?

No, and I think biotech is interested in this. There's very interesting ways to do this pharmacologically and so we may see Those mature faster CD38 inhibitors. Obviously there's a whole different financial structure there. If you can make a patent approved medicine, all that economic incentive is great for driving first science answers and then clinical answers.

What are the top labs right now in your mind in studying NAD and its precursors or ways to increase it?

I'm too much of an outsider to get into naming names on that, I'm going to only get myself in trouble, other than to credit Joe Bauer for being a fantastic collaborator on it.

Okay, let's pivot to the final thing I want to really get into, Josh, which is cancer metabolism. It kind of ties in so much of what we've talked about. And. And that's how you and I reconnected five or six years ago at a conference, and then obviously a number of times since then. So the irony of it is, right, you do your PhD in the inner workings of how the immune system works, but you're not particularly interested in cancer at the time. You come back to academia as a metabolomics expert, and now you've kind of wound your way back to oncology in a way. Tell me a little bit about that journey. Right. How did you go from this profound interest in metabolism, metabolomics and flexomics to realizing a beautiful application for this is in the field of oncology?

Part of it is really the human connection. I was so fortunate to be at Princeton, which is this kind of academic bubble where I could do my experiments on E. Coli and yeast and really set up these good metabolic measurements unmolested. Also, we're close to Pennsylvania. At some point, I got a call from the head of the Penn Cancer center at that point in time, Craig Thompson, saying he wanted to visit. Of course, I just say yes. That was kind of a life changing call for me because it brought me into the world of biomedicine again. Basically, in the context of working on cancer metabolism, it was very natural because if you look at the history of cancer therapy, first great rational triumph in treating cancer was antifolates and Sidney Farber name memorialized on the Dana Farber Cancer Center. Right. This is really the origins of how cancer was rationally treated by targeting metabolism. And it just got understudied for so many years. And it was a very natural re entry point for me because cancer you can study as isolated cells in a culture dish, much as we were studying E. Coli and yeast. And so that was much more comfortable to me in the 2008 or whatever time frame, trying to work on mice. Delighted that we got back to mice a few years after that.

What year did Craig leave Penn to go to Memorial Sloan Kettering? Must have been shortly after he invited you over. Right?

A few years after.

So people like Craig, who I have not had on the podcast, but I'd love to. But Lou, who I have. When you think about cancer metabolism today, I mean, it's just a Booming field, I would argue. And no disrespect to people in different fields, but cancer metabolism and immunotherapy are really two of the most promising and exciting areas in the field today. Which were two things we didn't have a single word about in medical school, right?

Yeah, that's absolutely true. Maybe some anti metabolites for cancer hidden somewhere in the pharmacology book. You know, one of the most exciting things is going to be the interface of those two fields. And we see this with microbiome composition being predictive of whether immunotherapy works. Amazing work from Jennifer Wargo showing that fiber can promote the effectiveness of immunotherapy. And so we're seeing that connection also being made. Soluble or insoluble tbd, there are different flavors of soluble fiber. And I have my pet dreams for how this may work mechanistically, but I think, like, it's going to be an incredibly important interface.

So tell folks a little bit about what it is about cancer cells that makes their metabolism distinct from their non cancer counterparts, but within the same tissue. Even like if you want to compare adenocarcinoma of the colon or breast or prostate and look at the perfectly normal non cancer cell sitting right next to it, we typically talk about two hallmarks of cancer, right? We talk about the inability to respond to cell cycle signaling. So this is why these silly things just keep growing, even when they're told stop growing, and then the capacity to metastasize, to basically pick up, leave, go, grow in a new site. But what is it about them metabolically that also is a piece of their signature?

Cancers tend to be glucose users once you step back. And in the fasted state in particular, using glucose is a weird thing rather than a default thing. The default thing is to use fat and lactate. Then the fact that cancer uses glucose is very distinctive. And they do this in large part because they're programmed internally to basically feel like they're always seeing insulin. And this is through mutations in something called the pi3 kinase pathway that Lou Cantley, who you mentioned, pioneered. And this leads to the fact they're positive on this FDG PET scan. So they'll constantly take up and phosphorylate and trap glucose or glucose analogs. And this is actually the most sensitive way to detect most types of cancer. Downstream from this, though, there's a ton of metabolic changes in the cancer cells. The most fundamental of these is the fact that in order to do the uncontrolled growth, they have to do uncontrolled nucleic acid synthesis. And this is the vulnerability that was targeted initially by farber, but it's been targeted by a lot of very important medications that are widely used still today. Pematrexat is first line treatment for lung cancer. And if you get these medications right, you can induce mutations through the metabolic stress on the nucleotide system. And this can make immunotherapy work better. That's a very exciting part. And I think that part has gotten understudied as cancer metabolism has returned turn to the fore. There's been a lot of focus on fuel usage cutoff, which is tough because like most cells in the body, cancer cells can use a lot of different types of fuels depending on what's available.

Yeah, this is an important point, Josh, because a lot of people, I think, would hear the first part of what you're saying, and their natural conclusion would be, well, wait a minute. If you do a pet scan on somebody and it lights up with glucose, that tells me cancer loves glucose. Ergo, the way to treat cancer, don't eat glucose. Problem with that logic is no matter how little glucose you eat, you still have plenty of glucose in your circulation. I mean, even if you're in a complete state of starvation, again, going back to George Cahill, 40 days of starvation, they still had 3 millimole of glucose in their circulation 40 days out. So there is no way to eliminate glucose. Now, an argument could be, but you're going to minimize insulin. So I guess the question becomes, is minimizing insulin actually more important than minimizing glucose? But the idea of starving cancer seems potentially overly simplistic. Right. Based on everything we've already talked about.

I think starving cancer is very, very hard. And as you say, getting circulating glucose to go meaningfully down below the healthy 89 where you last measured yourself is very, very difficult. Even if you could do that, it's not going to prevent the cancers from having access to internally stored fuel for a while in the form of glycogen, and then ultimately to amino acid fuel and fat fuel and lactate fuel, ketone body fuel. And we've shown very clearly that cancer can use all of those things. They're all valid inputs. They can't replace glucose in the test tube. But it's not easy to cut off the cancer fuel supply, Especially not without cutting off some other critical fuel supply, the immune cell fuel supply. Right. Which would be a disaster. Or the brain fuel supply, which would be an even more acute disaster.

This idea that there's a Way to exploit the metabolic. I don't want to say limitation, But I would just say quirk of cancer in a way that also augments the immune system. Say a bit more about that, because that's both fascinating intellectually, but also elegant in that it's mechanistically in line with a bias I have, which is cancer's really going to be hard to get under control. So hoping for a stalemate where you use multiple modes of action Is probably a better strategy Than hitting really hard on one lever. Again, it's a bias of mine, but at least I can acknowledge it.

I think we're seeing a lot of moves to try to make cancer into a chronic disease where the therapies are not so terrible. Hitting the nucleic acid side of cancer is something people are trying for maintenance therapy. And I We need to think about that whole side of cancer metabolism fresh, Because I think there are targets waiting to be developed. If they create nucleotide imbalances, which is a natural thing to do when you hit that system, then nucleotide imbalances are drivers of mutations, and mutations in cancer cells are drivers of immune response to cancer. So that's one very appealing avenue. Another.

So in other words, interrupt their ability to synthesize DNA, they will create more mutations. More mutations is more shots on goal for the immune system.

That's one really exciting avenue. Another exciting avenue is to apply a very strong stress to the cancer While putting pressure on their fuel supply. I think it's very hard to think that you're going to put so much pressure on the fuel supply that that alone is going to make the tumor slow or even more optimistically, regress or something. But if you come in with chemotherapy, for example, that's already targeted preferentially, not perfectly, to the tumor, and then you pair that with something like ketogenic diet, which is lowering insulin, lowering glucose, Then we at least see in animal models that this can be a very powerful combination. We see that the tumors start to deplete glucose in response to the chemotherapy. Whether that's because their vasculature is breaking down or whether that's because they have heightened glucose demand because they're having mitochondrial damage from the chemotherapy, I'm not sure. But we see that chemotherapy lowers glucose in the tumor intrinsically. And then if you come in with a diet that lowers glucose availability, this becomes stronger. And then you can get to really low tumor glucose. And we see pretty big improvements in outcome in mouse experiments. Hopefully they'll translate to the clinic. We have a clinical trial Open on this now.

And what's the best tool we have besides the conventional? And maybe it is simply the conventional in terms of ways to interfere with their nucleic acid synthesis. Is it literally just going back to old school chemotherapeutics that do that?

Well, for the moment, yes. I mean, for the moment, pematrexate is probably the most successful clinical agent. Gemcitabine, other things of this sort are all well used as part of the armamentarium. But I think we need to think fresh. It's really interesting to me that when we were in medical school, I thought we would not see a cure in our lifetimes for hepatitis.

And look at that now, huh?

And look at that. And that was mainly nucleoside analogues.

I mean, we were told that, because I remember talking about this as, why can't there be a vaccine for hep C? And it's like, you'll never be able to vaccinate a flavivirus. Okay, well, that still turns out to be true. But you'll never cure hep C. And, yeah, lo and behold, and this is.

Just nucleoside analogs as the centerpiece of this. And so the fact that there's clearly untapped potential there. Now, maybe that potential was maxed out 40 years ago when people were doing this hardcore for cancer from the cancer but not hepatitis perspective. But my guess is that that's all chemistry that's evolved a lot, and that this is a ripe area for rediscovery.

Well, it's interesting you mentioned gemcitabine because of course, that's one of the first line agents for pancreatic cancer. And if I'm not mistaken, you have a particularly keen clinical interest in pancreatic adenocarcinoma, is that correct?

Yeah, it's the cancer that I've worked on the most. It's obviously just a horrible disease.

It's definitely one of the cancers that gives cancer a bad name. It's the fourth leading cause of cancer death in both men and women, yet by incidence, it's a fraction of that. It just speaks to how lethal it is. You know, the last time I looked, Josh, I would say that adenocarcinoma of the pancreas is 95% lethal. And I've heard people argue that the 5% who don't die are misdiagnosed, almost suggesting that it's pretty much impossible to survive pancreatic adenocarcinoma, which is the worst thought in the world. So you certainly picked a tough one to study.

I feel A very strong commitment to it because of a bunch of reasons, but just the fact that it's so terrible is a motivation. And I think it was a disease that for a long time it's just so terrible we just give up. I don't think that's the right attitude for terrible diseases. And one of the hardest things in making biomedical progress is getting a clinical readout. And the hidden positive in how terrible this disease is is the clinical readout is just sitting there itching to be improved. There's the capacity to do really compelling clinical tests of any idea and it's a terrible disease. Most clinical efforts are going to fail, but they can be done relatively fast, relatively cost effectively. And we're seeing progress. Fulfuronox is progress. A lot of patients tumors respond even more will respond. If you combine two agents that were approved. Gemcitabine and abraxane is an albumin balm form of paclitaxel with a platinum agent. That triple combination produces regressions in most patients tumors.

But they're not durable. I mean, that's the thing that's killing us.

They're not durable. Okay, but the duration of response is terrible right now. But the fact there's response is promise. Normally, from my perspective, once you can start seeing response, you're on the road. We have to figure out how to make the response durable. I hope that's where the metabolic part becomes important. It's going to be some interface of the metabolic part or the immune part, or a yet harder hit with chemoradiotherapy earlier diagnosis. These are the hopes for fixing this.

Is there something about pancreatic adenocarcinoma that you've observed metabolically that is distinct from other gastrointestinal adenocarcinomas?

You know, colon is also a terrible disease. Liver is also a terrible disease. So all the gastrointestinal adenocarcinomas are unfortunately really bad diseases.

But at least with those stage one, certainly stage one colorectal cancer is survivable. Hepatocellular is a bit tougher, but you're better than a coin toss. But again, coming back to pancreatic stage one is 80 to 85% not survivable. I've always wondered what is it about pancreatic adenocarcinoma that is so difficult? And is it simply that its rate of early metastasis is so early that stage one is just sort of a misnomer term? There's no such thing as stage one.

I think that's a big part of it. It's kind Of a soft organ, the pancreas, A very invasive cancer. And you can have local invasion so many places from that site in the.

Body, plus immediate access to the portal system that's just seeding the liver constantly.

It's just seeding the liver. And so it's anatomically a really problematic location for keeping the cancer self contained. Metabolically, it's a very tricky cancer. It's almost solely driven by mutations of the ras oncogene. Not saying there aren't other drivers, but almost every patient has this ras driver. And this is an instruction manual for the cancer cells, not just to divide, but to do a bunch of metabolic things that involve scavenging nutrients from the environment and taking in nutrients in non standard ways. And so it actually instructs the cancer cells to reach out arms, pull in nutrients, internalize them, Degrade macromolecule nutrients from the environment, and use this as a garbage recycling form of nutrient access that makes them very metabolically pernicious. The other thing that we see that's really interesting in new work and mouse models of pancreatic cancer Is that they don't have to be very metabolically active in order to be horribly lethal. So the pancreas is a master protein producing organ. You may think of insulin as the most famous protein to come out of the pancreas. Right. But the bulk of the pancreas is not beta cells that make insulin. It's exocrine pancreas.

Yeah, that's only 5% of it. The exocrine is the real gland.

And the exocrine pancreas is just making digestive enzymes like crazy. It does by far the fastest protein synthesis in the body. The cancer turns that protein synthesis way down. So it's not hyper metabolic, it's just that it has this huge capacity to make stuff that even when it turns it down, still has enough biosynthetic capacity to grow and divide and grow and divide. Because it's turned down its main energy consuming normal function of protein synthesis. It can function with much reduced TCA activity, reduced ATP synthesis rates.

So it's very efficient then.

Very, very efficient. So it can turn down all these normal functions, and it still has the capacity to reproduce the cells and make these horribly invasive and metastatic cells, which are ultimately lethal.

Of all the epithelial cancers today, for which we don't have a cure, which is almost every one of them. Right. Shy of like a gist or something like that, or certain testicular cancers, Is there one that you're more optimistic about in terms of Metabolic approaches to therapy.

You see pematrexate being used effectively in lung cancer. I think you see the cancers with mutational burdens being the ones where you're getting the good immunotherapy responses, Whether they're ones that are particularly susceptible intrinsically to metabolic effects, I don't know. I don't think that's going to be the standalone heart of treating any of these cancers. I think it's more going to be a key piece of the puzzle in getting enough either drug killing by preventing their metabolic escape mechanisms, or enough, enough immune activity. And those may be opposites. So you may need also kind of cyclic therapies where you go through rounds of metabolic suppression in order to keep things calm while you can, and then periods of metabolic augmentation that are really directed at augmenting the immune system. I'm a big believer that, you know, there's metabolic limitations on immune response to cancer and that if we can overcome that, we will have major therapeutic benefits.

You mentioned ras in the context of pancreatic carcinoma. Ras is rarely immunogenic in the pancreas. It's a driver mutation, but doesn't give us a beautiful little 9 to 11amino acid peptide that gets presented on an MHC class molecule. Right. It's the great irony of this whole thing.

You need more shots on gall, you need more antigens.

Do you have any sense of how many tumor infiltrating lymphocytes are typically identified at all in resected pancreatic specimens?

It's typically quite the lymphocyte desert. There's a ton of macrophage activity in pancreatic cancer. And so I think macrophage rewiring is going to be a big part of allowing lymphocytes to enter. And these are areas where I think metabolism can be quite impactful.

Well, Josh, this is super interesting. I hate that we're ending on somewhat a depressing note. Is there anything more optimistic that we want to talk about than pancreatic adenocarcinoma beyond the statement that, hey, hey, look, this is why we have the smartest people working on the hardest problems. But anything else within cancer metabolism specifically that you think, boy, 10 years from now, I'm really optimistic that we're going to have a new way to hack into their DNA synthesis pathway in a corrupt manner that just spits out mutations using kind of novel systems. I don't know, Andy. Sens oligonucleotides, like just something that totally disrupts them in a selective manner.

My Big hope on this front is that we're going to be able to have some combination of directed metabolic immune supplements and diet that really work with therapy to treat cancer. I mean, cancer is such a discrete disease. The clinical trials are so manageable, and the fact that things are metabolically messed up up in the tumors, so incredibly clear, and they're so clearly messed up in a way that's favoring the wrong kind of immune cells. And so ultimately, through either some sort of supplement or diet, we're going to be able to reverse that and we're going to make immunotherapy work instead of for 10% of patients, for a majority of patients. So that's my uplifting thought for you, is that metabolism will be part, along with the, you know, better pure immunological therapies of getting immune control of cancer over the coming decade.

And besides reducing insulin, which is such an obvious strategy, are there other metabolic levers to pull with the diet? Because really, between ketogenic diets and cyclic fasting, those are kind of the two ways that you do that. Do you see evidence of amino acid restriction or any other nutrient restriction that could potentially play a role?

I think amino acids are complicated, but they hold a lot of potential. I think the type of fat can be important. Saturated and unsaturated fat are really different, and in cancer they're going to play, you know, different roles. So it's very nice work from Matt van der Heyden's lab showing that a higher saturated fat ketogenic diet could be more tumor suppressive in some contexts because the tumors have trouble making unsaturated fat in the context of hypoxia.

Say more about that. I wasn't aware of that. I know Matt's work very well, of course. Is he still at Dana Farber?

He's at the Koch at mit. So he's actually head of the Koch now, the MIT Cancer Institute.

And was this in prostate cancer?

This was. Oh, I forget what cancer background he did it in. I think it was in pancreatic, at least in part, but I'm not 100% sure.

So that's super interesting. So a ketogenic diet that was higher in saturated fat posed a greater problem for the cancer cells because they couldn't make, presumably the essential unsaturated fats.

Exactly. I think this is a interesting strategy. I mean, those effects were relatively subtle up to now, but, you know, it could be part of the picture. I think the really exciting part of the diet is also the parts that connect to the microbiome So I think the fiber part really working that out. And maybe total protein matters in ways that we don't understand and needs to be. Maybe we need to not just think about cycling, fasting and feeding, but cycling. You know, for example, there's a time maybe when you want to come in with a lot of carbs in the absence of protein, and that may achieve something that creates a particular immune milieu and then, you know, you need protein in the right timing after that. So there's a lot of things we can do with timing of macronutrients that can be interesting.

Seems like an eternity before we'd be ready to study this in a human clinical trial because the permutations are so many. So do you feel like we have high throughput animal models where we can test these hypotheses and say we've looked at 10 ways to do this in animals, but these are the three most promising, so we're going to kind of go ahead and do these now.

You know, the good animal models of cancer are still not that high throughput. And there's a lot of challenges converting animal diet and human diet. We'll come out with some work showing that, you know, some of the most exciting dietary combinations are absolutely effective in animals, but they're not effective through the mechanisms that people thought before. Because even in animals, trying to get the diets aligned so that you really isolate variables is tough. I think the fact that we're asking these questions that haven't been asked before is going to build momentum and we're going to build this interface out over the next five year period in animals and we'll do clinical work as a field in parallel with that. That has an impact and has an impact on patients lives within the five to ten year timeline.

I hope. Do you worry that the challenges of even if you came up with the right diet, so let's just assume 10 years from now, the answer is a cyclic ketogenic diet that has this much saturated fat, this much monounsaturated fat, this much polyunsaturated fat, this much glucose on this day, this much protein on that day, the formula exists. So this is almost an impossible thing, but that it's impossible to adhere to in the way that a pill or a drug or an infusion is much easier. Or do you think that that in cancer, because the stakes are so high, adherence will be unlike it is in any other field of medicine?

No, we have to make it simple. This has to be clinically actionable. I would go much more Back to the first question that you asked. Maybe when I first mentioned the potential of immunotherapy and fiber, like, is it soluble fiber, is it insoluble fiber? And then there really are different flavors of soluble fiber. Now, maybe it's the G of them, or maybe it's one in particular. Maybe it's one isolatable molecule that relates to that. And then we have one isolated molecule, tiny molecule, that will more than double the number of complete responses you get to immunotherapy in mice. One tiny molecule smaller than glucose.

You already have that.

We already have that as a metabolite.

So it could almost be nutritional supplements as opposed to wholesale dietary changes.

These can be supplements. The dietary changes may be in a very acute way. Just the way the patient comes in the hospital for a tough bout of chemotherapy or a tough surgery. Maybe we're going to go to a place where we take people's glucose in the hospital almost down to zero for 12 hours with a deep ketosis and some pharmacotherapy at the time that we hit them really hard with chemo and 24 hours of that. It's like night and day in terms of the overall effect. But it can't be asking patients to give up eating and giving up the joy of food or another trial that we're starting now is a trial with SGLT2 inhibitor plus a low carbohydrate but not fully ketogenic diet to see if it can put people in ketosis. And then just looking forward to would this be a convenient way to get the benefits of ketosis in cancer patients while still allowing them to have a little bit of breaking bread with the family?

Last question for you is totally random. I don't know what made me just think of this. Princeton is the only Ivy League school that doesn't have a medical school, correct?

I think that's right.

There's got to be a deliberate reason for that. Princeton is fantastic in everything. Do you know why it doesn't have a medical school? And is it just the proximity to Penn or. It was assumed that that's where the collaborations would be.

Princeton doesn't have a medical school because at its heart, Princeton is a hybrid of a college and a university. And it is an institution that has the ultimate priority under undergraduate education. It's committed to that. It's best in world in that. In assessing how to be best in world at undergraduate education, the Princeton administration many times has asked the question, is having medicine on campus part of that? And the answer has always been no. Let's have a somewhat more pure intellectual environment and keep our focus on doing the very best training for undergraduates.

It doesn't have a business school either, and I guess it's the same argument.

There's no business school, there's no law school. That's Princeton. It makes it very special and it's good to have special places that are distinct. I think that's a wonderful thing.

Are you at all a fan of Richard Feynman's work?

At a very light level, I guess I would say that's the best way to put it.

There aren't places around Princeton that you go to see what eating club he was in or things like that. You don't look for the old places.

I read a giant biography of Oppenheimer and the film of that on campus. So Matt Damon was apparently on campus. I'm guessing he's playing Oppenheimer, but I haven't checked anyway, so he has to become a small Oppenheimer fan after reading that intensive book.

I have three of Feynman's books, meaning books that were actually his. So I have his table of integrals from when he was in high school and then two more advanced calculus books, one from when he was at Princeton and then one from when he was his first professorship at Cornell. It's just. They're sacred to me. Right? It's like his scribblings, his notes all over these things signed Richard P. Feynman and what his address was and things like that. I've never made the pilgrimage to look for his eating club and things like that. It probably doesn't even exist anymore, but. Just wondered if you'd been on the tour.

No, but you should come down. There definitely have been some awesome Princetonians. I guess my kids all went to nursery school in the building where Von Neumann built the first computer. So it is amazing the amount of stuff that happened around here.

Well, Josh, so great to see you again. I hope it's not too long before I see you in person again, but really appreciate it. First of all, the amazing work that you've done over the past 20 plus years and sitting down to share it with us today.

It's been fun. Appreciate the opportunity.

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 the subscription. Now to that end, membership benefits include a bunch of things. One Totally kick ass comprehensive podcast show notes that 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 Ask Me Anything episodes. Hearing these episodes completely access to our private podcast feed that allows you to hear everything without having to listen to spiels like this the Qualys, which are a super short podcast 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 to learn more and access these member only benefits, you can head over to peterattiamd.com subscribe. You can find me on Twitter, Instagram and Facebook, all with the ID Peteratti 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 and does not constitute the practice of medicine, nursing or other professional healthcare 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 PeterAttiamD.com about where I keep an up to date and active list of such companies.

Sam.