#66 - Vamsi Mootha, M.D.: Aging, type 2 diabetes, cancer, Alzheimer's disease, and Parkinson's disease – do all roads lead to mitochondria?

Foreign. Hey everyone. Welcome to the Peter Attia Drive. I'm your host, Peter Attia. The Drive is a result of my hunger for optimizing performance, health, longevity, critical thinking, along with a few other obsessions along the way. I've spent the last several years working with some of the most successful, top performing individuals in the world, and this podcast is my attempt to synthesize what I've learned along the way to help you live a higher quality, more fulfilling life. If you enjoy this podcast, you can find more information on today's episode and other topics@peterattiamd.com. Hey everybody. Welcome to this week's episode of the Drive. I'd like to take a couple of minutes to talk about why we don't run ads on this podcast and why instead we've chosen to rely entirely on listener support. If you're listening to this, you probably already know, but the two things I care most about professionally are how to live longer and how to live better. 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Again, the podcast will remain free to all, but my hope is that many of you will find enough value in 1 the podcast itself and 2 the additional content exclusive for members to support us at a level that makes sense for you. I want to thank you for taking a moment to listen to this. If you learn from and find value in the content I produce, please consider supporting us directly by signing up for a monthly subscription. My guest this week is Dr. Vamsi Mutha. Vamsi is a professor of Systems biology at the Harvard Medical School. He has an appointment at the Broad Institute, which is actually where we met, to conduct this interview, and we talk a little bit about what makes the Broad so special. He specializes in rare mitochondrial diseases as opposed to longevity per se, something that I love to talk about, but I think you'll see in this interview why it's so interesting to talk to someone who specializes in rare orphan mitochondrial diseases about longevity. His laboratory uses a blend of genomics, computational biology, biochemical physiology, and systems biology to study mitochondrial function and dysfunction. He received his bachelor's in mathematics and computer science at Stanford before going on to Harvard as part of the Joint MIT Harvard program. In medical school, he stayed in Boston to do his training in internal medicine, though as we discussed, he now focuses exclusively on research. He's received more honors and awards than I could name here, but it's always worth mentioning it when someone is a genius award recipient. So he won the MacArthur Foundation Award in 2004, which obviously puts him in pretty rarefied Earth. In this episode, we talk about a lot of things. We start with, at least for me, one of the best discussions I've ever had on the mitochondria. And you might think at first that some of this recapitulates things that were discussed on previous podcasts. You may recall we had a great discussion on the mitochondria with Navdeep Chandelier, but we go a little bit deeper and we talk more about some of the evolutionary pressure around the mitochondria. And even though at first you might think, well, gosh, this seems awfully scientific, where's the application? If you stick with it, you're going to see where it comes and how understanding these rare orphan diseases that most of us have never heard of can give us an insight into aging. We do eventually start to talk about metformin, which I know many people ask about, and he provides a great insight into, or several great insights into what metformin may or may not be doing. Again, at least for me, this was like just the master's class in mitochondrial biogenesis, electron transport chain, et cetera. Talk about ways that we can target mitochondrial proteins and complexes to treat disease. But perhaps the single most insightful and interesting thing that we talked about that completely blew my mind was the role of hypoxia as a treatment. I want to repeat that again. The role of hypoxia oxygen deprivation as treatment. Now, we're going to cover this in a way that I think is very interesting, so I don't want to say any more about it, but I do want to add a disclaimer to this that Vamsi and I spoke about after the podcast, and I want to make sure it's here right. So VC emphasizes that their published research to date on the beneficial effects of hypoxia in animal models of mitochondrial disease is still in its early stages and it's restricted to animal studies. It is not yet ready to be applied to humans. To extend these ideas to humans would be premature and irresponsible, since hypoxia can have life threatening implications. If and when the concept is extended into humans, it will need to be done so in a clinical trial setting with the appropriate ethical, regulatory and safety measures in place. So with that caveat and without further delay, please enjoy my conversation with Dr. Vamsi Muthka Vemsey. Thank you so much for making time today.

Oh, absolutely. Welcome.

Yeah, yeah. Sight unseen, too. I'm always impressed when people I don't know in advance agree to sit down with me. I'm kind of honored, Humboldt, so. So thank you very much.

Thank you for coming and meeting.

Yeah, the Broad is. Is quite an impressive place. Tell folks a little bit about where we are right now and what makes this unique, even within the hallowed halls of Boston biomedical research institutes.

No, I think it's a really exciting place. And I've had the pleasure of actually watching it blossom from nothing. It' the brainchild of somebody named Eric Lander, who was one of the leaders of the human genome sequencing project. And as that was being completed in draft form in 2001 and then in quote, finished form in 2003, he saw the need to create some sort of an entity that would take advantage of the power of genomics for improving biomedicine. So he created this institute, and it's unprecedented in a lot of ways. It's joint between Harvard and mit, it involves all of the hospitals here in Boston, and it's basically a incredible forum where people can get together and basically pursue projects that they can't pursue on their own in their own individual laboratories. There's a big computational theme here. There's a big theme on being systematic in one's approach and not focused narrowly on the protein that they've studied in the past, but being systematic and seeing where the data takes you. And there's a very pervasive theme of collaboration as well. So Eric has always said that the Broad Institute is a bit of an experiment, but now it's been about 15, 16 years, and I think, without a doubt, it's been a wildly successful experiment.

Do most of you here at the Broad have an appointment elsewhere? Like, I know you spend much of your time at Mass General. Lots of people here spend, you know, they have an appointment at mit. Does everybody have another appointment outside of the Broad?

Almost. So there's almost two types of people that work at the Broad. There's some people that are actually formally employed by the Broad Institute Institute, and there's others like myself that are employed elsewhere. In my case, I work at Mass General Hospital and Howard Hughes Medical Institute. That's my primary appointment. My paycheck comes from there. But then I spend a day, a week over here working on collaborative types of projects that I can't pursue easily in my own lab. So there's almost two types of folks over here. I think one of the neat things about the Broad Institute is traditionally there's this grad student, postdoc assistant professor, academic track. But how about for all the other people that want to do research in a nonprofit academic setting but aren't interested in applying for R1 grants or teaching? The Broad actually has an entire research scientist track as well. And so you can be employed here as a research scientist, doing science and.

Funded here and funded here.

And I think that's a very, very different organizational model compared to any other academic organization.

Let's back up for a moment. You studied at Stanford where you. You did. Were you a computer science major? What was your major in undergrad?

I was math and computer science.

Okay, got it. Did you know you wanted to get into biology and medicine?

You know, when I was a kid, I'm an Indian American. My father is a retired surgeon. As it turns out, my three older siblings would end up becoming doctors also. So I like to joke that we had the medicine gene in our family. So growing up, I was convinced I wanted to be a doctor of some sort. But then in high school, I fell in love with math. I did the high school math competitions, was pretty good at those, did the high school science fair competitions in math, did well with those. And so I ended up going to Stanford with the goal of being a math and computer science major. And that's what I was squarely focused on. And then towards the end of college, when I was looking for a research project, my advisor told me about the work of Sam Carlin. He's a statistician that was developing some of the underlying methods for biomolecular sequence analysis. He's a fundamental mathematician and statistics.

And this is like early 90s, early 90s.

That's right. That's right. So I ended up working on DNA sequence analysis as a college student, writing code to try to analyze DNA and protein sequences. And I fell in love with that. And then my advisor said, why don't you contemplate a future career in medicine if for no other reason. Going to medical school is a great way of learning physiology. I thought about it from a practical perspective. I applied simultaneously to PhD programs in mathematical biology and also applied to MD PhD programs. And then I applied to this program joint between Harvard and MIT. I actually thought that it was an MD PhD program. This is all pre Internet. So I thought it was an MD PhD program, but it wasn't. It was a straight MD program, but it's a joint program between the two institutions.

Yeah, they would Set aside, like, I remember, like 20 students for this. What was it called? The HST. HST program.

That's exactly right. So I was a part of the HST program and it was kind of catered to students that had slightly quantitative backgrounds, math, computer science, types of backgrounds. So it provided a bit of a more gentle introduction to medical school.

I could have used that program. My first year of medical school was just unbearable. I mean, it was such a culture shock of. I don't know if I told the story on the podcast before. The worst exam I've ever done in my life, from the point when I decided to care about school, was the first semester histology exam in medical school. Because I very arrogantly and naively assumed that if I understood the concepts of histology, I didn't need to memorize anything. So I took this very pure math approach, which was I could just derive everything in my mind if I understood the fundamentals. And that got me a 53% on the final exam in histology, which, like, I just sat there looking at this number thinking, how is this possible? Am I the stupidest human being that has ever walked? The face of it was really a wake up call that said, you know, you're going to actually just have to start memorizing things around here.

It's a big culture shock, I think, when I know you have a background in engineering and applied math, so very similar to my background. And it made for a lot of unhappiness for me also. That first semester of medical school, at least you were in California. I was facing the Boston winter at the same time I was facing histology.

That's a good point. I did have that going for me. So when you finished med school, you did a residency in internal medicine. You stayed in Boston, Correct. You stayed at the Brigham.

That's right. That's right. I've been here now for. It's hard to believe, but about 25.

Years during residency, the Brigham is obviously certainly among the top three most academic medicine programs in the country. Was talking to one of your colleagues yesterday and, you know, he made the point that when he looked at sort of his class or the, you know, the cohort of people that entered medicine at the Brigham, you know, you just fast forward 20 years and they're basically the leaders within each of the different scientific fields. So I assume that was a pretty deliberate decision on your part to really preserve sort of academic optionality.

You know what I think at almost every single one of these junctures, the nodes. Yeah, yeah, exactly. From college to medical school, medical school, to residency. I think maybe I'm just inherently a bit of an indecisive person, but I was unsure as to whether or not I wanted to do a residency. By then I'd fallen in love with basic research and the question was, was I going to do a basic science postdoc or was I going to do a residency? And so just as I did at the previous node, I decided to just apply for both. So I simultaneously applied for postdoc positions and for residency programs. That was the era when you would fill out your residency match list with a number two pencil. So I'd fill it out every day.

The Dixon Taikana Roga number two pencil. I love those things.

So I would fill it out and then I would rip it up the next day. I'd fill it out and rip it up the next day. And I joke that the only reason I did a residency is because the due date was an even number.

So that said, I want to put you on the spot and ask you a question that I get asked all the time and frankly, I don't know the answer to it and I feel bad that I always provide sort of nebulous answers. But I do get asked a lot by either college students who are contemplating medicine or not, or medical students who are interested in research. And the question is a variant of one, if you're at the college node, but you're very interested in medical research, is there benefit to doing an md? And then the second order question is, if you're in medical school and you're going to finish and you know you want to do research, is there a benefit to doing a residency? Now, I generally tell people, and if you disagree with me, I hope you say so very loudly. I generally say, if you're in college but undecided about medical school or not, I would say don't do it. Pursue the PhD. If you are in medical school and presumably at least somewhat interested in medicine, still do an internal medicine residency, because there is no substitute for doing that type of research and at least having the ability to understand clinically why you're doing it. So again, do you disagree with that? And if so, how would you or how would you modify that?

I also get asked this question quite a bit because I think there's a group of people out there that love science, are probably good at clinical medicine if they do it, but they're also good at research if they do it. And maybe they're inherently a little bit risk averse as well, so they're always trying to figure out what's the best path. For me, the advice that I had gotten when I was in college was that the reason to go to medical school if you're interested in research, is that it's one of the best curricula for understanding physiology. How do all of the parts come together and operate and trying to understand how an entire living system operates. If you don't go through all of medical school, if you don't learn anatomy, if you don't learn histology, if you don't learn cardiovascular physiology, it's a bit tough.

And so it's a good point you make. Right. Which is in somewhere between 16 and 24 months, which is the pre clinical part of medical school, it's almost impossible to get a more well curated view of the human body because it's been optimized for that over 100 years.

That's exactly right.

More.

And it's a living system, right? I mean, you could study yeast, you could study Drosophila, but the human is extraordinarily well investigated. And you're right. In about two years or so you have a very intense curriculum studying one living system, different aspects of it. So from that perspective, I think it's a great education. It's a broad education for biomedical research. At the other node, again, I spent a lot of time thinking about this and one of my advisors actually said, at least do an internship because then you'll be licensed, you can prescribe medicines at that point. And there is something special about caring for patients. There's something very special about going through the process of residency with your colleagues. And again, you really get to see the human living system at some of its extremes. So I think it's a great way of continuing to learn physiology and pharmacology as well. So I'm super grateful for the path, of course, and of one experiment. But I'm really happy that I did medical school. And I'm also super happy I did the clinical training as well because it completely shaped my research focus as well.

Okay, no, that makes sense. I generally say the same thing, which is I don't regret the path I took, though I'm glad I didn't know in advance what it was going to look like because it would have seemed too indirect, but nevertheless. So right now you spend virtually all of your time doing research, is that correct?

That's right.

When did you stop seeing patients?

It's been about five to six years or so.

Was that a tough decision?

It was tough because we went through this entire medical school and residency process with the goal of actually seeing patients and caring for them. The truth is, the number of patients I was seeing was asymptoting as a function of time. Emotionally, it was very, very difficult to go to zero. So even though I was seeing probably one patient a month or so five years ago or so, going to zero was actually the harder part. But at some point I had to just do the math and say there's only a certain number of hours per day. I'm running a pretty big research group that's focused on mitochondria and mitochondrial disorders. The long term goal is to impact these patients. And so I'm going to let other people be the ones that care for these patients at the front line, and I'm going to try to develop new diagnostics and drugs for these patients.

So let's start talking about the mitochondria. When did you come to the realization that that was the area you wanted to focus on? And what is it about the mitochondria that especially drew you in?

So as a first year medical student, I was a little bit unhappy here in Boston. It was cold. There's a lot of memorization. That first year, that first semester in particular, I wasn't sure if I had actually made the right decision in coming to medical school from a math background. And then right in the middle of that first semester, when we were taking our histology and pathology class, we had a very, very brief lecture on myopathies muscle diseases. And there's one slide that basically indicated that there's a rare form of myopathies that are due to mutations in the mitochondrial DNA. Now, truth be told, I don't think I'd appreciate it at that time that we had our own mitochondria with their own genomes. And so that immediately fascinated me. You dig a little bit deeper, you learn that these were once bacteria. They're kind of swimming around in our cell. So that's kind of cool. You see an image of mitochondria in the muscle. It's just beautiful. There's just something that really captivated me and I kind of got hooked on that organelle that first semester of medical school. And I've told this story to other people, Peter, but what happened was a few weeks later, my dad's cousin, who is at that time a postdoctoral fellow in the Harvard Research, one of the research hospitals, she heard that I was unhappy. So she invited me over to her house in Somerville for a Friday night dinner. So it started to snow. I took the red line all the way to Somerville. It was a 20 minute walk to her house. By the time I had gotten in, my shoes were soaked, I was freezing. She immediately looked at me and said, oh my God, you look terrible. And so she took my coat off, she tried to dry me up, and she started cooking dinner. And then her boyfriend appeared like an hour later. And now it's about 8 o' clock or 9 o' clock or so. We had dinner together and then we found out that the T had been shut down. And so not only was I an unhappy first year medical student, but now I'm spending Friday night with my dad's cousin and her boyfriend in Somerville. And so they create a makeshift bed for me in their living room. I'm spending the night there at this point and I look at the bookshelf and there's a textbook of mitochondrial biology on the bookshelf. So just a few weeks earlier, I had this initial encounter with the organelle. I saw this book, so I just took it off the shelf and I just started reading it. I read about 100 pages that first night. I borrowed it. And then I basically devoured that book over the course of the next few weeks. And I just fell in love with the organelle at that point and thought to myself that this is what I want to work on for the rest of my career.

That's an amazing story. And I think for people listening, they shouldn't be concerned that they haven't had that eureka moment. Right. I mean, I've heard a lot of amazing scientists talk about their passion and not all of them. In fact, most of them don't have such a laser moment like that. Now, of course, the stories of people that have those moments are even more vivid in my mind of that. Oh my God, I can't think of anything but this. So let's talk a little bit about the mitochondria. You've already alluded to some of the really interesting features about it. So let's start with the. Well, gosh, which one's the most interesting? Let's just start with the lineage. Right, so how did these bacteria find their way into these eukaryotic cells?

Yeah, this is, I think, one of the most fascinating aspects of the organelle. So this is a process known as endosymbiosis. So the current theory is that there is an organism probably resembling a modern day gram negative rod, something like a rickettsial species that merge.

Folks might not even know what that means. So gram negative is just a staining that allows us to identify a type of bacteria. So you've got this bacteria that is shaped like a rod, literally, and how does it get its energy? Prior to this encounter, the thinking is.

That it had a full electron transport chain and was probably capable of doing aerobic metabolism using things like oxygen as a terminal electron acceptor. And one of the theories proposes that that had an electron transport chain that's not that different than modern day mitochondria. The hypothesis is that that somehow merged with something resembling modern day archaea. So there's three main domains of life. You have archaea, you have bacteria, and then you have eukaryotes. And eukaryotes have nuclei. So the hypothesis is that there's an archaeal species, there's a bacterial species, they form a union of some sort, and this is what gave rise to modern day eukaryotic cells, maybe about one and a half billion years ago.

That's kind of remarkable and is the idea, I mean, what kind of evolutionary pressure must have been placed to create an entirely new species? It's also binary. It seems somewhat binary to me. Is it. In other words, when you look at the difference between, I don't know, say, a chimpanzee and an ape, or an ape and a human, you can see lots of continuous evolution. You know, Neanderthal, et cetera, et cetera. What you're describing sounds much more switched on, switched off. Is that the case? Was this a. Or were there lots of iterations that we can't even appreciate today in between the union and the current paradigm?

As far as we know, endosymbiosis of the mitochondrion only took place once.

Wow.

So there are lots of eukaryotes. Almost. Not all, but almost all eukaryotes have a mitochondrion. And if you look at the DNA of those mitochondria that still have a genome, the phylogenetic analysis tells us that this was a monophyletic event. This took place only once. Now, that's not true for something like the chloroplast that also arose through serial endosymbiosis, and that likely took place at least two independent times in evolution. But for the mitochondrion, it probably only took place once, as far as we know.

And the chloroplast, of course, is to a plant, effectively what the mitochondria is to an animal like us.

That's exactly right, yeah.

So how many genes do we think that that gram negative rod had?

Great question. Probably about 1,000 to 2,000.

And at the time, the Species that it merged with would have had how many? To the best of our guessing, probably.

Also a few thousand.

So you had two things that had comparable numbers of genes merge and yet today you or I would have 30,000, 20,000 genes inside the nucleus and we'd have, is it 13 genes inside our mitochondria? 13 preserved genes. So in other words, to a first order approximation, the mitochondria lost all of its genes. But a deeper dig says actually it somehow hung onto 13. Why do you think that was?

This is what we call reductive evolution. Modern day mitochondria actually represent a mosaic. So you need about 1,000 proteins total to make our mitochondria. And so some of those are attributable to the original bacterial ancestor and others are brand new innovations that even that original bacteria did not have. But on the reductive side, approximately 1,000 genes from that original bacteria have either been lost altogether or have been transferred to the nuclear genome. So that that genome today is tiny. It's only about 16,000 bases. It encodes 13 proteins. But that's only if you're looking at animals. There's a lot of different eukaryotes, so there's a lot of mitochondrial diversity. So you and I still retain 13 proteins that are encoded by our mitochondrial genomes. But if you look at malaria, it's also a eukaryote, it has a mitochondrion, but its genome only encodes three proteins today. So that's additional reductive evolution.

What about things like, you know, flies, yeast, Are they also variable?

Flies are also animals. And so most animals, you would put.

Them in the category.

Yeah, that's right. So they would have about 13, sometimes 14 proteins. But if you look at something like Giardia, which causes a terrible diarrheal illness, beaver fever, that's a eukaryote, it's actually lost its mitochondrial DNA altogether.

I know we talked about 13 proteins. Is it one to one mapping or are there genes that are non coding or is it.

Right, so the mitochondrial genome encodes two ribosomal RNAs, 22 TRNAs, and then 13.

Thirteen proteins. Yep. So obviously to have 13, we pretty much have a good sense of what the function of each of these are. How many diseases that afflict humans result from genetic disorders there inherited mutations that produce dysfunctional proteins.

Right. There's about 250 different syndromes of the MTDNA of the mitochondrial DNA. And by syndrome I mean there's a particular mutation that's associated with a particular clinical phenotype. But that's only if we're talking about the mitochondrial disorders of the mtdna.

That's right. Is that a higher or lower frequency on a probability basis given the number of base pairs? Because you said something like only, what, 16,000 base pairs, it's relatively tiny. We have genes in our nuclear genome that are 10 times that size. So from a probability basis that you could have 250 mutations in that 16,000 base pair that would each lead to these distinct mitochondrial diseases, is that more or less robust from a DNA perspective than our nuclear DNA?

I don't think we have a good answer to that question. There's a little bit of an ascertainment bias in clinical medicine. So when I was going through residency, probably when you were also going through your clinical training, the answer to almost every single question about mitochondrial disease was maternal inheritance. That's because the nuclear genome was sequenced in draft format in 2001, but the mitochondrial DNA was sequenced in 1981, so that was almost exactly 20 years earlier. And it's small. And so as soon as you saw patients that had a particular clinical phenotype that could be a mitochondrial disease, it was easy to sequence the mitochondrial DNA. So beginning in 1988, when two papers were published reporting the first mutations in the mitochondrial DNA, it's been relatively straightforward to sequence it and associate mutations in the MTDNA with the disease phenotype. This is why now there's about 250 of these MTDNA mutation clinical phenotype mappings. The nuclear genome has been a little bit slower to follow since 2001, beginning even a little bit before the completion of the genome sequence. We've now as a community, been able to identify about 300 different genes in the nuclear genome that underlie mitochondrial disorders. And of course that's 300 different genes, but then there's different allelic variants as well. So I think it's going to be a little while before we can answer your question in a satisfactory way. But it's a really provocative question.

And one more number to sort of extract from that is do we have a sense of how many genes in the nuclear genome are required for mitochondrial function? So in other words, what's the denominator, nuclear wise?

Right. That's actually one of the areas that we focused on heavily in our laboratory. So beginning soon after the sequencing of the human genome, we knew that the human genome encodes about 22,000 proteins. So an important question is which of those find their way to the mitochondrion. It's Going to be more than 13. These are elaborate organelles. So we used a lot of methods in the early 2000s, things like proteomics, GFP tagging, microscopy, computation. And we're able to identify about 1100 proteins that are made by the nuclear genome that find their way into the mitochondrion. So those 1100 proteins have to work with those 13 proteins in space and time to do all the amazing things that the organelle does.

How do they communicate? Do we. Do we know how expression of MTDNA is coordinated with nuclear DNA?

It's a really, really interesting question. So some of the mechanisms are known, but a lot of mechanisms are not yet known. So when you exercise, for example, if you're deconditioned and if you do a combination of aerobic training and strength training, you can actually increase the number of mitochondria. And there's an entire transcriptional program that will turn on all of those nuclear genes. But that same program will also turn on the replication factors that will go into the organelle and cause the mitochondrial DNA to replicate as well. So it's a really smart transcriptional program that says, in response to exercise, make more of the nuclear encoded components and make more of the mitochondrial DNA, and make more of the mitochondrial DNA encoded proteins.

So that increases mitochondrial density in a given cell. So you have a myocyte, a muscle cell. Do we know what the actual signals are? They basically instruct the cell to make more DNA. So what is the input from the exercise? You mentioned two types of exercise, right? Strength training and aerobic activity. Those have very different physiologic properties. So what is it that at the physiologic level is leading to this nuclear level?

One of the important signals is something called AMP kinase, the change in the ATP to ADP ratio. That's known to be one of the activators of this particular transcriptional program that induces mitochondrial biogenesis.

So this would be a decrease in.

AMPK activity, An increase in ampk.

An increase.

Okay, so that is basically sensing something.

Like the ATP to ADP ratio going down. Yeah.

So when we exercise, the ATP levels are usually pretty nicely defended. But then what's happening is there's another reaction that's taking two ATP molecules, making an ATP liberating amp. While ATP levels are defended, some of these ratios change, and that can be sensed. And that's one of the inputs into this program that says, let's make more mitochondria.

Now, we'll come back to this far down the line, but just because you mention it one of the effects of a drug called metformin that everybody loves to ask about is it activates ampk. Does that imply that metformin administration by itself alters mitochondrial density?

So I think when we talk about exercise, I think AMP activation is generally regarded as one of the important signals for mitochondrial biogenesis. But I don't think it is sufficient.

It's not sufficient. It's necessary, though, potentially, I think it is.

But I would need to review some of the older literature to really confirm that.

So what do you think some of the other signals are?

I think some of the other signals are things like calcium. And clearly there's other signals. There's a couple of disease states as well, where we see a massive proliferation of mitochondria. They're not functioning properly, but there's a massive proliferation of malfunctioning mitochondria. So we're trying to work out some of those signals as well. So, as of right now, it's a bit of an open question as to exactly how the number of mitochondria is sensed and regulated. But we know some of the inputs, right?

We know crudely what the inputs are. Right. Exercise, as you mentioned, what are some of the other things that even hormonally, for example, did nutrients play a role?

One of the studies that I was a part of about 10 to 15 years ago or so show that even things like androgens and testosterone can actually influence the amount of mitochondria. Disuse is a great way of rapidly eliminating mitochondria. And then in terms of nutrients, NAD is an important signal as well. This transcriptional regulator that I'm talking about, it's called PGC1 Alpha, and upstream of it is something called SIRT1, which of course utilizes Ned as a cofactor. So there's a couple of these inputs.

I'm having lunch with David Sinclair later today, so we'll be talking plenty about that.

Absolutely, absolutely. So I think a couple of these signals are beginning to emerge because we're not at that stage right now where in a pill we can put seven of these things, give it to a patient, and boom, we can replace exercise. We're not there yet, but it'd be remarkable if we understood the process well enough so that we could one day.

Yeah, there's been lots of claims of exercise in a pill. I remember the New York Times wrote about something called Irisin. God, they wrote about it in 2011. 12. And then I saw it again recently, and. And it's funny, of course, you See these stories which you realize they're being written about in such a crude way that you can infer nothing. So you go back and look at the paper and you realize it's kind of a ridiculous claim at this point. What is the turnover of mitochondria in a cell? So if you have. Let's put some scale to things for folks. So I'm going to take, I'm going to biopsy one cell from your quadricep, one muscle cell. How many mitochondria approximately would be in it? Assuming you're a relatively well conditioned individual.

Talking about the number of mitochondria is a little bit ambiguous because they're not quantal units. Mitochondria will constantly undergo fusion and fission. So at any given state you can have a different number of mitochondria. The mitochondrial DNA is a quantal unit. So you can ask how many mitochondrial genomes are there per nuclear genome in a cell type? Okay, so a fibroblast will have a few hundred, maybe a thousand copies of the mitochondrial genome for each of each.

Copy of its nuclear genome.

That's right. But there's a lot of variation.

The highest would be what, like a cardiac myocyte or something?

Or a neuron, the unfertilized egg. Wow. It has half a million copies of mitochondrial DNA.

See, I wouldn't have guessed that. I don't know why. I could have come up with 10 guesses and that would not have been one of them.

It's remarkable. And dad's sperm probably has a few hundred at best.

And I would have guessed the sperm needed more because it's doing the motion right. It has to fight to get to the eg.

Part of the reason is that the egg is so big relative to the size of the sperm. So that's to a first order approximation, the explanatory variable. But the unfertilized egg is sort of the Olympic gold champion when it comes to mtn.

It's the Michael Phelps of mtdna.

That's right, that's right. And then red blood cells of course have no mitochondrial DNA.

Then what's the turnover look like? Right, so if you have a cardiac myocyte that presumably lasts for a long time, you're in an individual. Like these cells are not turning over quickly. Are they turning over new mitochondria constantly? How often is that mitochondrial DNA churning over? I guess what I'm trying to get at is what's like the half life of these sort of non discrete mitochondria.

I think in most non Dividing tissues, the half life is on the order of a few days.

Wow. So we are cranking out mitochondria.

That's right. That's right. There's differences between dividing cells versus non dividing cells. When you have a dividing cell, obviously as a function of the eukaryotic cell cycle, you need to double the number of mitochondria and then you partition that into two, then you double partition. So dividing cells have very different mitochondrial turnover dynamics than non dividing tissues like muscle or neurons.

There's like a hundred other questions I want to ask, but I also don't want to. I want to get us back to the beginning so that we can set the framework to ask some of these other questions. So you alluded to something, right? Which was, I believe, something that, at least for me, until very recently, I didn't think was challenged or questioned, which was the origin of said mitochondria being maternal. So presumably in the early 80s, when we first we, like I had anything to do with it. When people far smarter than me sequenced mitochondrial DNA, given how few the number of genes were, it was not difficult to realize, hey, this all seems to come from the maternal side, not the paternal side. And as recently as several years ago, that was considered almost an axiom. All the DNA in your mitochondria comes from your mother, not your father. Has that been called into question lately? Have there been exceptions to that?

I think the textbook teaching is that the mitochondrial DNA is transmitted exclusively maternally. And the reason for that is related.

To this comes back to the egg, I'm sure, Right?

It gets back to the sexual dimorphism. The egg is huge. It has half a million copies of mtdna. Dad's sperm is tiny. It only has a few hundred copies of mtdna. So by sheer dilution, it's very difficult for dad's MTDNA to get transmitted. Right? It's outnumbered half a million to 100. But on top of that, there's actually active mechanisms that will seek and destroy dad's mitochondria. So the mitochondria coming from the sperm are coated with a protein called ubiquitin. So after fertilization, those mitochondria are actually actively eliminated by a surveillance program. So not only is it very difficult for dad's DNA MTDNA to compete with a huge number of MTDNA molecules from mom, but those that do make it inside are actively destroyed as well. So mechanistically, these are the two reasons why MTDNA is passed on almost exclusively from mom to child. Now, with that said about 15 years ago or so, there's a case report in the New England Journal of Medicine that took a biopsy of a particular individual and they saw MTDNA molecules with two different haplotypes. And then they looked at the haplotype of the father and then they concluded that this was a rare case of paternal transmission. So that paper from about 15 years ago was sort of the lone exception to this rule that we accept as an axiom. And then about a year ago or so in 2018, another paper emerged again claiming that in a few families there is paternal transmission of MtDNA. It's a rare event, but this is an active area of research. Rules are made to be broken. And I think two things. Number one, I think other people will have to try to replicate these results to make sure that, that even if they're rare, they're real and not some sort of a technical artifact. And then number two, if they're not technical artifacts, I think there's an opportunity to learn something very, very deep about the mechanisms of maternal transmission.

Right. Because that would suggest, based on the explanation for how this takes place, it's hard to deny that the first one's going to continue to take place, which is just the stochastic sampling, but you could certainly see scenarios under which the find and kill program malfunctions and therefore you sneak in a little bit of the paternal DNA.

That's right. And in fact, in one of these papers that was just published in 2018, they speculated that perhaps there's a mutation on the nuclear genome in that program. In that program, so that it's possible for dad's MTDNA to get transmitted.

Well, that is interesting, but I think to the first order approximation, we can still assume that mitochondrial DNA is maternal.

Yes.

So let's talk a little bit about sort of Mitochondria 101. So you're a first year medical student learning about the mitochondria. Here's basically what you learn, right? You've got this inner membrane, this outer membrane. I forget what the term is. They call it the powerhouse of the cell or something like that. I think there's some sort of glib term that I know that people who study mitochondria, like yourself and Navdeep Chandel, who I've interviewed, sort of bristle at the simplicity of such a term. But the idea is, all things being equal, a cell takes in glucose free fatty acid substrate for energy. And let's start with the glucose, because we'll probably spend more time talking about glucose Today, through what I can't even remember, 10 steps, we turn glucose into pyruvate, more or less. We then basically have a choice. A cell has a choice based on the availability of oxygen and the rate at which ATP is being demanded. So if oxygen is scarce relative to ATP demand, you can take a inefficient route, but at least you guarantee to get some ATP, which is you can turn the pyruvate into lactate. And lactate itself is interesting, and maybe we can talk about it, but you generate some ATP. If the demand for ATP is not as great and there's sufficient cellular oxygen, you can take that pyruvate and make acetyl CoA. And that acetyl COA then becomes one of the substrates leading into this thing called the electron transport chain you alluded to. Walk us through what happens in that latter scenario.

I like to think of the mitochondria as being the key place where there's energy transformations. So in order for a cell to work, it needs energy. But in the same way that in order to live our lives, we need to have one type of battery for our iPhone, a different type of a battery for a laptop, a different type of a battery for our automobile. We need energy packaged in different ways. So this is sort of the charm of the mitochondrion. What it does is it's going to take fats and carbohydrates and proteins, and it's going to break it up, almost like a Cuisinart. And as it's breaking, breaking it up, it's going to harness the electrons. And that's what's called an electromotive force. That's one type of energy. So you're probably familiar with things like the NADH to NAD ratio. That's basically an electron carrier. So that's one form of energy. And certain types of enzymes can be powered directly by NADH and nad, but then that can also be converted to a gradient, a voltage across the inner membrane, a different type of an energy form. And that energy form can be used to drive transport.

So let's explain what that means to people. So everybody knows what a battery looks like. Like literally a Duracell battery that you stick into whatever, you know, your kid's toy. And that's typically about 1.5 volts, right? A 9 volt battery gets its name because it has 9 volts, but everyone recognizes it as the goofy square one. We take this, I think, for granted because we sort of have these backgrounds in math or engineering and stuff. But I think for the average person. It's helpful to understand what voltage means. And you just alluded to it, right? It's a potential, it's a gradient that is created by disproportionate placement of electrons. And it's only with that that you can generate power. So why does a battery die? Why is it that Pretend I'm still using a Walkman, I put my two 1.5 volt AA batteries in my Walkman, at some point it stops working.

Why at some point it's not going to be able to hold charge. And so I'm probably more familiar with the mitochondrial battery than I am with the Duracell battery. But when you have nicely functioning mitochondria, you can charge them. You can create a nice voltage that can be used for work, you can dissipate it, you can recharge it. But at some point when it gets older, when the membrane is a little bit leakier, when it can't, it'll stop holding charge.

So it's that ability to keep a difference in charge across the membranes that is the same reason your little 1.5 volt battery in your Walkman. I love that I'm saying Walkman, by the way, there's half the people listening to this won't actually know what that means. Or the 9 volt battery in your smoke detector. Or frankly, the fancy. I don't even know how many volts a Tesla battery is. It's like 12 volts, I'm guessing, or something like that in an electric vehicle, whatever. But there's a reason you have to keep charging these. But at some point, even when you charge them, they cease to work if it's a rechargeable. And you're basically saying, look, think of a mitochondria as partially being a battery.

That's right. That's only one form of energy. Right.

So that's taking chemical to electric.

That's exactly right. And so you can have an electromotive force, you can have, have a proton motor force, and then you can dissipate that battery to basically catalyze the formation of ATP, which most people know is the energy form that's used to power muscle. When you exercise, the mitochondrion is doing all of these elaborate energy transformations from electrical potentials to proton potentials to phosphorylation potentials and different enzymes and, and processes and machines in your cells will use one or the other.

This is so exciting. Like, I've tried to have this discussion with my daughter, who's 10 and she's not Quite at the point where she sees why. I think this is amazing. But she's almost at that point where I guess you can look at a piece of food on the plate and explain why eating that is essential. Where is the energy in that food? So maybe we'll use this as sort of the example to go full cycle. So you are looking at a Cheerio on your plate right now. That Cheerio is mostly carbohydrates, so we'll simplify this. And it's got glucose in it, it's probably got more complex carbohydrates in it. But at a molecular level, it's a lot of carbons joined to hydrogens, carbons joined to carbons, and carbons joined to oxygens and oxygens joined to hydrogens. That's probably most of the bonds. Correct. So bonds contain energy. There's chemical energy there. Which of those bonds would be the most energetic? Probably the carbon hydrogen in total number, just given the ubiquity of it. Right. A carbon hydrogen has more energy than a carbon carbon. I'm guessing.

I'm not too sure.

I think that's the case. In the show notes, we'll list what the potential energy is in each of those bonds. But nevertheless, you go through this process of actually eating the thing. You put it in your mouth, you break it down mechanically. You've broken it by the time it exits your stomach. But it's really once it gets absorbed out of the bloodstream that you begin this chemical process of breaking those bonds. And then you get something for free. Right. When you break those bonds, that's when you're getting the energy to create this electron gradient.

That's right.

Which you then use at the end to basically do this. One thing you alluded to, which is phosphorylate the ATP. So that energy gradient allows you to then put a phosphate back onto an adp. That's the most obvious one from food. Now, you talked about NAD and nadh. Can you say a little bit more about what those are and how they fit into this? In particular, in the mitochondria?

Again, the classical teaching is that NAD is an electron carrier. When you have two electrons, it's going to be in what's called the NADH form. And if you don't have those two electrons, it's in the oxidized form, which is the NAD form. That's another way of holding the energy that can catalyze reactions. What we're learning over the last few years is that in addition to this role as an electron carrier, The NAD itself can be used as a substrate.

For other reactions outside of the mitochondria.

Or inside, outside of its role as an electron carrier.

Okay. And that biggest role for that is in these. I guess we'll allude to it, but there are these complexes in the mitochondria.

But that still as a redox carrier. So the electrons go from the nadh, are transferred to the electron transport chain, and that's basically a wire that's going to conduct the electron until they hit oxygen and make a water. That's a downhill process. And during that downhill process, these protons are pumped across the inner Membrane, generating about 150 millivolts. And that can then be used to do work including catalyzing the conversion of ADP to ATP. And that's what we call oxidative phosphorylation.

Yep. But then talk about the other example that you were using there.

And this is a newer area of biomedicine, and actually David Sinclair has worked in this area quite a bit. But everything that I just told you relates to electrons coming on and off of nad. So when we talk about the NAD to NAD ratio, that's sort of the redox potential. But that NAD molecule, it can also participate as a substrate in certain chemical reactions.

So sirtuins use nad.

Exactly, sirtuins. Some of the DNA damage response pathways, the parps, they'll actually use the NAD as a cofactor. So that's important because it's possible in certain states, like when you have a damaged cell, for example, if you have a cell with damaged DNA, that NAD can decline very, very rapidly. It's an electron carrier, so you're actually losing the ability to carry some of this charge.

Now, this is interesting, right? Because observations are, as we age, these NAD levels decline. Is that due to greater demand for it? Is it due to a reduction in production? And of course, the clinical question that everybody asks is, is there benefit to replacing it?

What's really interesting is there's a couple of different signatures of the aging process. So if you biopsy muscle from individuals of varying ages, you'll see a gradual decline in the NAD content. If you quantify the amount of mitochondria using any of the different metrics, you'll see a decline. If you look at things like VO2 max and skeletal muscle as a function of age, you'll see a gradual decline. There's this gradual decline in NAD and in mitochondrial activity as a function of age. I think the big question in the field is, do you just have an old and sick tissue. So you have sick mitochondria, or will targeting the mitochondria actually somehow alleviate age associated decline in tissue function?

This is such an interesting question and something that probably until a year ago, I don't think I spent enough time thinking about, which is what does it mean to age at the level of the mitochondria and what are the implications of it? And perhaps most importantly, what can be done to slow the rate of aging? Now you study a problem at sort of a different node, which is you are looking very specifically at diseases that people, most people haven't actually heard of. And I don't want to say you're not interested in those diseases per se, because you are, but they're basically a gateway for something else. So later on in this podcast, I suspect we'll come back to more of this. Mitochondrial fitness, health, inflammation, mitophagy. There are many other topics I want to explore with you, but let's now, having laid the groundwork, go back and talk about your work and what you're learning. So give me an example of some of the diseases that you study in your lab.

We've historically placed a lot of emphasis on a very large collection of individually rare inborn errors of mitochondrial metabolism. These are typically single gene disorders. They can be due to recessive mutations in the nuclear genome, or they can be due to mutations in the mitochondrial genome. But at the end of the day, there's a component of the mitochondrion that's defective at birth. And so what we just spoke about is the fact that as all of us age, there's a gradual decline in the activity of mitochondria. The big question in the field is whether that's cause or consequence. These other 300 rare monogenic disorders of mitochondria, there's no doubt, there's no question.

The gene did the randomization for you. You know, cause and effect.

That's exactly right. The mitochondrion is defective at birth. And now we can actually evaluate what the consequences are.

You said about 300. What is the phenotypic spectrum? How many of these, for example, are fatal within the first year of life.

They tend to follow a bimodal distribution. The recessive mutations in the nuclear genome, they tend to present early in infancy, within the first few weeks or months of life. The mutations in the mitochondrial genome, those tend to present a little bit later in life.

Wow. So let's focus on the latter group for a moment. I mean, unless you'd prefer to start with the former. Which one do you spend more time looking at.

We spend a little bit more time on the nuclear genomes. Okay.

Because they present earlier and presumably they're more severe.

That's right.

Okay, so give me an example of what some of those mutations are and what their phenotype is.

One of the clinical syndromes that we study is something called Li syndrome. So there's about 80 different genes that can be mutated to give rise to this clinical syndrome, which is basically a subacute degeneration of gray matter. It's a very rapid neurodegeneration. It's a terrible, terrible disease.

And what age are people when they start to experience this neurodegeneration?

So most of these kids are actually born developmentally. Okay. And then within the first couple of months of life, there's some sort of a stressor. Sometimes it's some sort of an infection, sometimes it's dehydration. That'll put them into a neurometabolic crisis. And at that point, if you look at their brain MRIs, you'll see lesions in the brain stem, the basal ganglia, sometimes the spinal cord, corresponding to regions of necrosis.

Wow. So quickly Fatal. And what did you learn? I mean, you have one syndrome, but there are many paths that produce it. Are there common threads to the genetic insults that lead to this awful phenotype?

No. Great question. That's exactly what we're trying to figure out. So thanks to genetics, we've now been able to. We as a community, I mean, have been able to map out genes in the nuclear genome. Some of these are in the mtdna, but at the end, we get this thing called LI syndrome, and we're trying to figure out what exactly is it about the broken mitochondrion that gives rise to this phenotype. And honestly, we don't know what the full answer is right now, but it's a very, very active area of research right now.

It might be naive, but just listening to you describe this, I can't help but think, can we learn something about Alzheimer's disease or other forms of neurodegeneration, which I think many people are starting to argue are basically neuronal energy crises? So there are lots of insults, right? You can have an accumulation of toxin, you can have a insulin resistance, frankly, you can have microvascular disease. All of these things are predisposing people to neurodegeneration. And something that they could all have in common is depletion of energy to the neuron, which would be perhaps the most sensitive cell to an Energy reduction. I mean, anybody can think about that for a moment. If you know somebody who's lost their ability to breathe for a period of time, usually the thing we care about the most is their brain, because that's the first thing that you suffer from when you have a hypoxic event. So is that based on Lee syndrome? Is that the explanation for why you're potentially seeing it disproportionately in the brain versus skeletal muscle? Is it just the sensitivity of the brain to energy withdrawal? Or do you think there's something specific about the mitochondria in neurons?

Mitochondrial disorders can actually impact almost any organ system. And so Leigh syndrome represents one type of clinical manifestation of mitochondrial disease, but there's another set of disorders that impact the skeletal muscle as well. I think at this point, we don't know why. Mutations in one subunit of the electron transport chain gives rise to brain disease. A mutation in the neighboring subunit of the same protein complex that's equally evolutionarily conserved will give rise to muscle disease. Wow. There's a massive non linearity over here that we simply don't understand right now.

So if there is one and only one, silver lining in these awful diseases, it's that scientists will have no shortage of questions to ask for decades to come.

No, I think this is a super active area of research right now, in part fueled by the link between mitochondrial decline and aging. So that's such a complicated problem. You know, trying to understand why a car breaks down after being in service for 25 years. It's hard. Is it? Did some fan belt break? Did the battery stop charging? Did the tire deflate? It's kind of hard to know why an entire car breaks down after 25 years in these rare mito disorders. We have 300 different forms of that automobile that was almost broken at birth, if that makes sense. We study them in part because these patients need new treatments and therapies. And so that's enough of a motivation for us. But we also do expect that a subset of them, by studying them, will inform what's happening in the more common form of aging.

Your lab studies oxygen, but not necessarily in the way that most people commonly think about it. Most people, when they think of oxygen and mitochondria, something that comes to mind pretty quickly is Ross.

Absolutely.

I spoke with a friend of mine, Navdeep Chandel, and we spoke a lot about Ross, and now I've had a great take on it, which was, look, we think of RAs typically only in the negative, and they do lots of Negative things. But they may also be a signaling molecule and therefore an essential thing. Talk to me about the lens through which your group looks at oxygen.

So remember a while ago, you asked a very, very astute question. You have these 80 different genes. When they're mutated, they give rise to this thing called Li syndrome, which is a different type of a neurodegeneration. What does that pathology, pathogenesis look like? The traditional dogma for mitochondrial pathogenesis is that when the powerhouse of the cell is broken, there's not enough ATP and there's a power failure. That's the traditional dogma. And without a doubt there's truth to that in some instances. What we've discovered is that in addition to producing ATP, mitochondria are also consumers of oxygen. Most of the oxygen that you breathe, Peter, is being consumed by your mitochondria. When a patient has a birth defect in the mitochondrion, in addition to not being able to produce sufficient ATP, they also have excess oxygen as well. So oxygen delivery tends to be patent in these patients, but the utilization ends up being poor.

And oxygen just asking for people to understand, how does oxygen even get to the mitochondria? So we all understand that we're breathing air that has oxygen. And let's even go one step further and just take it for granted that there's a gradient in the lung that allows oxygen to get into hemoglobin to a red blood cell. Now that you have a fully loaded red blood cell in an arteriole that enters a capillary, how many things have to happen for oxygen to get into the mitochondria specifically, not just the cell.

Oxygen is not particularly soluble in water or fluids. And so we have an oxygen carrying protein called hemoglobin that's found in our red blood cells. And so in the lungs, all of the red blood cells and their hemoglobin get loaded with oxygen. And now these red blood cells are delivered to peripheral tissues and the oxygen gets extracted from the fluid. And as the fluid gets depleted in oxygen, the hemoglobin will basically offload its oxygen. And so there's certain tissues that become extremely, extremely hypoxic. So the oxygen will get extracted by a tissue largely by diffusion. And the mitochondrion is basically consuming most of this oxygen.

So a cell that is sitting there doesn't require an active transporter to get oxygen across its outer membrane, so it just diffuses across. And then when oxygen enters the cytoplasm, how does it get over to the mitochondria?

Depending on the cell type, it either diffuses.

How does it know that the mitochondria is the place it needs to go?

Well, it's being consumed, so it's a little bit of a sink, basically.

Ah, so that's what I want to understand. There are lots of places oxygen could hang out. So what is the force that is drawing it into the mitochondria? Is it simply utilization that basically creates a vacuum?

That's right. That's right. Now, in certain tissues like skeletal muscle and heart, we have other oxygen carriers in the tissue. Things like myoglobin are oxygen carriers. So they're almost little buffers of oxygen that are.

Yeah, they're more like loaders, right?

That's right.

I think of them as like another storage for oxygen inside. Yeah, that's right. But that still has to get off the myoglobin and get sucked into the mitochondria effectively, right?

That's right. So it's another little storehouse, if you will. It's another buffer of oxygen so that when you're exercising, for example, you may not want to be oxygen limited, so you have a little extra oxygen in your myoglobin, but basically your mitochondrion is where most of your oxygen is being consumed. So it's a sink. And so that's the reason that we have gradients inside of our cells.

It's amazing. I've never really even thought about it this way, but it's sort of interesting to think at how efficiently the body disposes of carbon dioxide, because as you alluded to earlier, the end of that downhill gradient is a final path of electron acceptance, generating H2O and CO2, both of which we manage to largely off gas. So somehow those things have to exit the mitochondria, weasel out of the cell, cross the gradient and go back to. Well, in the case of CO2, get back to the hemoglobin molecule and get carried back. Like, it's like there's a lot of things going on here.

Well, so one of the things that we're discovering by studying these rare diseases, and this happened because of a CRISPR screen that we did a couple of years ago. But we've. What we've now discovered is that one of the consequences of mitochondrial dysfunction is excess unused oxygen.

So in other words, if a mitochondrion is failing to do its job, you will be failing to utilize oxygen, therefore you would see an excess accumulation of oxygen.

That's right. And we believe this is our hypothesis now, it's that some of that Excess unused oxygen is what is contributing to the pathology that we see in some of these rare diseases. It's a very, very different type of an idea. It's not all about the ATP. It's about excess unused oxygen. And we're not necessarily invoking reactive oxygen.

Yeah, I was just about to say, is it through free radicals, or is it actually oxygen to oxygen? No extra electron oxygen?

This is dioxygen toxicity that we're talking about. The way that I like to think about it is that if you have an automobile that's outside and it's rusting. It's rusting in part because the oxygen is directly oxidizing iron, you may produce a radical, but that's not the phenomenon. So the car outside is not rusting because of too much superoxide or hydrogen peroxide. It's direct oxidation of iron centers by oxygen. And so one of our hypotheses is that enzymes are tuned to operate within a particular oxygen range. And when the mitochondrion is not functioning properly, the oxygen levels rise. That excess dioxygen can now oxidize enzymes that will damage them as a consequence. So it's a very, very different way of thinking about mitochondrial pathogenesis.

Now, why doesn't the body correct for that? And note that while the oxygen in the mitochondria is not being utilized as quickly as my evolutionary prediction would allow, but therefore, we're gonna. There's less gradient pressure, so I'm gonna off gas. I'm gonna offload less oxygen to the cell with subsequent trips through. Like, in other words, you almost think this would have been corrected in a few milliseconds.

Well, I think in a healthy human, that's exactly right. We have pretty solid matching of oxygen delivery and oxygen utilization. But we're talking about patients that have broken mitochondria. So this is one of the things that we're actually trying to investigate. One of the ways that Ron Haller at the University of Texas Southwestern Medical center has proposed diagnosing patients with mitochondrial disease is to put them on a treadmill, measure their oxygen extraction. And patients with mitochondrial myopathies will often have high venous oxygen.

Let's explain that to people, because we're going to talk about VO2 max. And you've alluded to. So most people associate that test with sort of peak athletic performance. But let's talk about what it looks like. If I came into your lab and you were doing this, you'd hook me up to a device, you'd put me on a treadmill. You'd make me have to do some work to stress the system. You'd plug my nose and put a little miserable device in my mouth, basically creating a seal that would prevent me from being able to get oxygen or dispose of carbon dioxide in any place other than the gas chamber that is attached to. To the tube going into my mouth. You'd put me at the. You know, you'd ramp up the speed of the treadmill and you would be measuring essentially two things, the amount of oxygen you're putting in, and if it's room air, we sort of know what that is, but more importantly, the concentration of oxygen coming out. And that difference is what you're talking about. It's that extraction. Now, what happens in a normal person when you do this?

So, Peter, we actually don't do these.

Types of studies in humans.

And so.

But when one does this. Yeah, what would normally happen is a. What would you be measuring as a normal person works harder and harder in the difference between provided oxygen and returned oxygen.

So, again, we don't do these types of studies, but in these types of physiological studies that people like Ron Howell or other cardiologists will perform, they'll look at cardiac output as well as the oxygen tensions on the arterial and venous sides. And so by looking at all of these numbers, you can actually figure out quantitatively the number of O2 molecules being delivered as well as those that are being extracted versus those that are being returned to the venous system.

So you sort of do a ma. You can do a mass balance on oxygen, effectively.

That's exactly right. That's exactly right. And as it turns out, in these patients with inherited mitochondrial disease, for some reason the cardiac output is high, the extraction is low. In a healthy individual, usually the homeostasis is such that you're not going to be delivering more oxygen than you really need. So that homeostasis is somehow broken in these patients with.

So that patient would actually have a very high lactate level as well. Because if they're able to produce the cardiac output, but they're not doing it with oxidative phosphorylation, they're using their escape valve. So they're going to disproportionately have high lactate levels relative to a healthy individual.

That's right. So Ron Heller would actually argue that a high lactate in combination with a high venous oxygen is suggestive of a mitochondrial myopathy.

There's a researcher at the University of Colorado who is looking into this very phenomenon as an early indicator of type 2 diabetes. And it's really fascinating. I'm going to be going out there to spend some time with them this summer. They've made the observation that in the early stages of insulin resistance, the muscle, the skeletal muscle in particular, becomes inefficient at ox phos. So you start to see, even at baseline, even a person sitting at rest, their lactate levels could be twice as high as that of a fit individual. And of course, he came to this through the thinking that if you want to understand disease of the mitochondria, look at the exact opposite. Look at the fittest people in the world, look at the endurance athletes and ask the question, what do their mitochondria do? Well, and then what becomes the polar opposite of that in disease? Now, what you're describing is the most extreme example I've ever heard of this, right?

Absolutely. What you're describing is actually very consistent with a series of papers that were published probably in 2003 and 2000, including by myself, in collaboration with Leif Group and David Eltshuler, Ron Kahn, who used to be the president of the Joslyn, as well as Jerry Shulman from Yale, and Sri Kumar Nair at Mayo Clinic. All of us had sort of papers at the same time.

David Altshuler's here, right?

David Altschuler used to be here. He's now at Vertex, but he's one of the founders of the Broad Institute. But all of us had papers published at about the same time that show that if you take skeletal muscle from pre diabetics, healthy individuals that have a family history of diabetes but are still healthy, they'll all have a reduced number of mitochondria. The expression of those 1,000 genes required for mitochondria, it's just a little bit lower. If you look at any one gene, it's not significant, but if you look at the entire pathway, the entire pathway is down. The VO2 Max is down. So there's a series of papers back in 2003 and 2004 that led to the mitochondrial hypothesis for type 2 diabetes. The big question still to this date, almost 15, 16 years later, is, is that actually causal for the diabetes or is it just an early epiphenomenon? There's something else, X that lies upstream of the amount of mitochondria and independently the predisposition to diabetes, or is that a part of the causal path? And that's still unanswered to this date.

And there's nothing in a Mendelian randomization that can answer that question.

So as you probably know, these types of methods are only now becoming possible. So we're trying to work on those types of projects right now. You need large numbers of heavily genotyped individuals. You also need to have nice biomarkers or proxies for mitochondrial function. So those are some of the types of things we're trying now because it.

Comes up from time to time. Do you mind just explaining how Mendelian randomization works and why it's so powerful?

Again, this is not an area of my expertise. Others are much more expert at it than I am. But the analogy that they'll often use in describing a Mendelian randomness randomization is a little bit like a drug trial. So in a drug trial, what you'll do is you'll take individuals and you'll randomize them either to a drug arm or a placebo arm, and then you'll look for an outcome. There's an intervention which is the assignment of the drug or the placebo, and then you can actually check to see whether the outcome is correlated to that particular intervention. And if you see an effect, now that effect is attributable to that intervention.

Because the decision to give the intervention versus the placebo was done randomly. If you didn't do it randomly, you can't make that assertion.

That's right. That's right. Now, in a Mendelian randomization experiment, the idea is that there's a randomization that took place at birth. And so if you look at something like LDL levels, for example, an important question is, are LDL levels causal for heart attack, or are they simply correlative for heart attack? So thanks to genetics, we have a lot of genetic variants that can help explain the population variation in LDL levels once you have a good solid genetic instrument. Now, what we can do is we can take a very large number of individuals and we can actually draw a bell curve for what their genetic LDL level levels must look like. We haven't measured LDL in them, but from the genetics, you can actually come up with a bell curve in the population. And now you can say, let's take this tail, they've been randomized to a high genetic level of LDL versus this tail, a low genetic level of ldl. Now let's see what the outcomes are. And based on the statistics, based on the patterns of correlation, you can then make the indication inference that the LDL is either causal for heart attack or there's no evidence for causality.

I am not an Mr. Mendelian randomization jock, but My recollection in reading some of the papers about this is the one place that either as an investigator or a consumer of this science, where you have to force yourself to look closely is you can be fooled if the randomization of genes, meaning if the genes that you're looking at can also control something else that is related to the disease, that could have a counterbalancing effect. Is that correct? Is that like that's, that's one place where one has to be quite cautious.

I think there's multiple places where, I mean, as soon as you have feedback loops, you can get phenomenon called reverse causation. You can have your genetic instrument also has to be really, really. So again, this is not my area of expertise, so it has to be pursued with care, for sure. So there's probably going to be a fair number of false positive results for all true positive results from Mendelian randomization studies.

Yeah. But it is interesting. I would love to see the. Mr. When it's done for this particular question because again, you know, the implications are significant, both from the standpoint of preventing chronic disease or risk reduction in chronic disease, but also as we try to approach the question, the way that you phrased it a moment ago, which I really liked, which is everybody kind of has the gestalt of that car that just sort of breaks down and sometimes it's attributable to a catastrophic failure. Sometimes you blow the head gasket in the car and it's that it dies at that moment and it just becomes economically not feasible to put a new head gasket on. Other times it just gets harder and harder to start until it's just not worth driving anymore. So taking this to humans, how important is mitochondrial health to that process? In other words, does it become more often than not one of the drivers of this feeling, this lack of robustness, this lack of stability within the organism? And it seems to me that it's on the short list of candidates, Right? I mean, when this process goes awry, you are interrupting one of the more fundamental systems in human biology that affects almost every cell in the body, right?

Absolutely. I think it kind of cuts both ways because I think when other processes in the cell fail, I think the mitochondrion is at risk for also failing. So it's a highly reactive organelle as well, if that makes sense. It's going to be a tough question to fully answer. And this is why we like these rare genetic disorders, because they teach us about the pathology, pathogenesis of the organelle in a few defined Modes. And the big question for our field is, which of those rare diseases, which of those rare forms of pathogenesis bears any relevance to the common form of wear and tear aging? There may be a small subset of rare diseases where the fan belt is broken at birth. But it may be the case that the fan belt, as it turns out, is so resilient you never have to change it. And it's rarely the cause of a car breaking down. But it could be the case that it's actually the spark plug, right? It could be the case that there are certain birth defects where the spark plug is actually not working at birth. And as it turns out, with wear and tear aging, spark plugs have to be replaced. And so we have 300 different forms of monogenic mitochondrial disease.

Right? You have 300 single point of failures to study. And that gives you a beautiful picture of what it looks like.

Some of these are going to bear no relevance to the common form of aging. That's just going to be a fact. But the hope is that a small number of these will bear some relevance to the common form of aging. And just to be clear, we care about these diseases just because these are terrible diseases and we need therapies for them. And so that alone is motivation for our work in this area. Area. But it's also our hope that studying some of them will provide insights into the common form of aging as well.

You talked earlier about signatures of time, and another one of these signatures of time is inflammation within muscle cells. There was a paper that came out about a year ago that used STING to basically block the ability of a cell to sense breakdown in mitochondrial DNA. Are you familiar with this work?

I'm a little bit familiar with the STING pathway.

My understanding it's been about a year since I read this, maybe a bit less. Was that serially? If you study muscles as they age, you see more and more inflammation. So the question is, what is drawing inflammatory cells into muscle as we age? And the hypothesis was going back to something you talked about earlier. The DNA of mitochondria is bacterial in origin, and therefore, if you have a loose fragment of nuclear DNA in the cell, it shouldn't be especially immunogenic. But if you have a loose fragment of bacterial DNA in a cell that should actually be quite immunogenic, our immune systems would think of that as foreign. So the hypothesis was, what if the increase in inflammation we see is due to greater and greater mitochondrial damage that is leading to more and more exposure of mitochondrial DNA? And I believe, to test that, it used STING and remind me, I think STING actually blocks the ability of the cell to sense the mitochondrial DNA. Is that correct?

Not just mitochondrial DNA, but I think it's a general nucleic acid sensor and so there could be different sources.

I think it could block the sensing of DNA, period.

I think ordinarily it's designed to sense the DNA of incoming viruses or incoming pathogens, but you could imagine that if a mitochondrion ruptures that same DNA, nucleic acids can also be sensed. And you're right, because the mitochondrion used to be a bacteria. A lot of the components inside of our mitochondria resemble that of modern day bacteria and can be very immunogenic. I don't know if you follow. There's a study that came out of the Beth Israel Deaconess a few years ago on sterile crush injury. And as a former surgeon, you probably appreciate it. But the observation was that when you had an enclosed crush injury, so the skin has not been violated, there's a massive inflammatory response. So this investigator was actually trying to figure out what the inflammatory nidus was, did a lot of fractionation and ultimately figured out that there was some mitochondrial derived molecules. And I believe that they found that, that the mitochondrial DNA, as well as something called formylated peptides, are highly inflammatory. So remember those 13 proteins that are made by the MTDNA? The translation of those 13 proteins resembles the translation of bacterial proteins. And bacterial protein translation doesn't begin with a methionyl tRNA, it begins with a formal methionyl tRNA. Remember this F MET peptide?

Yeah, yeah.

This is.

My God, I feel like I'm back in medical school just for no other reason than for me, me to remember that. Let's go through that. So your DNA makes your messenger rna, your messenger RNA then moves over to be translated and to actually have the amino acids put in place to resemble the code that's being said. So talk about where the meth terminal end shows up there in you and I.

In eukaryotes, protein translation begins with a methionine residue. In bacteria, it's not a simple methionine, but it's a modified molecule. It's called formylmethionine and first order approximation, it's almost a signature of a bacterial derived protein. And we actually have receptors that are designed to detect formulated peptides. It's a sign of some sort of an infection. As it turns out, our mitochondria have bacterial origins, as we have discussed earlier. So these 13 proteins, they still begin with the Formal methionine.

Presumably, they can get away with it under normal circumstances because they reside within the mitochondria. They're protected and shielded from the immune system.

That's right. So in certain injuries. Right. This can actually escape, and it can basically activate the same inflammatory pathways that we have that are ordinarily designed to detect incoming pathogens.

I can't wait to actually find that paper. So that was. Who is the author on that?

I forget his name, but he's from.

The Beth Israel Deaconess Surgeon 0203ish.

No, no, no. This was probably within the last about five years or so.

Okay, well, we'll find that paper and link to it. But basically, that paper and the paper I was referring to from about a year ago, which I think was in science, both point to a similar conclusion, which is you can have a profound inflammatory response by simply damaging the mitochondria. And both of them would point to consistent, plausible explanations, which are the body confusing the contents for bacterial contents.

I think it's a very reasonable hypothesis.

Which then begs the question, if we believe that the aging inflammation phenotype is not beneficial, how do we prevent mitochondrial breakdown as we age? I mean, this becomes one of the key aging questions. Right. If you believe that. And again, I don't know what the best analogy is, but spark plug failure plays a role more often than not in the overall picture of decline. The longer you can protect those things, preserve those things, the better.

Another way at evaluating the causality question is if we had a drug that could somehow rejuvenate mitochondria, then you could ask the question, does directly intervening on this organelle retard the aging process? And unfortunately, as of right now, we don't have that type of a magic bullet. But exercise is one of the best ways of turning over bad mitochondria and inducing the biogenesis of good mitochondria. But the challenge, of course, is exercise.

Does lots of things. Yeah. Now, that said, we always. This is one of the differences between, I think, being a scientist and a doctor. When you're wearing your doctor hat, you just have to know what to do for that patient in that moment. And it's a luxury to know how and why it works. Right. So when we think about the importance of exercise, I've always found it ironic that I probably classify or qualify as an exercise addiction, like, in the true, unhealthy sense of the word. You know, I probably meet all the criteria of addiction and all of that stuff. But up until recently, I don't Think I really appreciated the value of exercise. I think it was honestly just something I did out of my neurotic pathology. But I actually, I think if asked, would have said that nutrition played a much greater role in health, sleep played a much greater role in health, and then exercise, you know, I mean, it's great, but you know, if you. I'd rather you eat well than exercise a lot or something to that effect, I'm certainly revisiting that. And of course, I also find it silly to do these sort of zero sum games. Like it has to be one or the other. You know, presumably doing all of these things well is the optimal strategy. But the deeper I look at exercise, and I'd love to know your framework for this because I'm still trying to create one. I'm putting exercise very loosely into three buckets. Strength training, very specific aerobic training. So this would be maximum mitochondrial output with minimal generation of lactate, and then anaerobic training, where you are basically demanding ATP at such a rate that you are really running through that lactate production pathway. Do you think that's a reasonable framework of buckets of exercise? Do you divide them even more granularly as you think about it mechanistically?

We're really not exercise physiologists. I don't think I can comment in anything.

Just personally, do you think of it in a way like that?

I think that's very reasonable, you know, just in hearing you talk. I mean, again, we're not aging researchers, but. So I'll ask you a question, if that's okay. Has anyone actually, you know, people have given metformin to mice, They've given rapamycin to mice. But has anyone given mice those three flavors of exercise to determine what the impact is on longevity?

Yeah, I believe those experiments have been done and I believe all of them show benefit. I'd have to go back and look at the literature in mice. In fact, I'm in the process of just starting to work on that chapter in a book that I'm writing. The problem is I generally bias against heavy mice literature, but you at least have the advantage of control. So the short answer is definitely each of those as a medication. Right. If you think of each of those as a pill, each of those produce a longevity phenotype. Where it gets challenging, I think, in humans is, well, I think there are so many ways to die when you get old that, for example, accidental death would rank in the top five causes of death for people over the age of 60. Now, the type of accident can change around. But by the time you're in your eighth or ninth decade, falling becomes such a significant cause of death due to the frailty of the individual that some of that exercise, for example, strength training, almost assuredly offers protection from that type of death. So the question is, I think it's a little hard to tease out how much of that benefit is in the cardiometabolic side versus otherwise. The other thing that's really challenging in studying humans is we don't have really good prospective studies in anything that resembles a longevity phenotype. So you are now stuck using something I think I recently described as the most servile trash ever shat into civilization, which is epidemiologic questionnaires to try to impute based on you telling me how you've exercised over the past 10 years, how that's going to predict your longevity phenotype. And again, the problem there is the dose matters, the specificity, the quantity, the quality, these things matter. And they're very difficult to tease out from these retrospective views. So I think the evidence is very compelling that exercise matters. And that's maybe less the question I'm interested in. I think what I'd love to gain insights into, and we may have to rely on non human models is just as we now can tailor a drug to do something very specific, can we tailor our exercise to be as optimal as possible? So if you took an individual who said, peter, look, I'm willing to exercise five hours a week, or I'm willing to exercise 10 hours a week, but I'm not going to be a professional athlete, how do I take those five hours a week or ten hours a week or whatever it is, and make the best use of it to impact all causes of mortality, meaning reduction of the risk of atherosclerotic disease, cancer, neoplasm, neurodegenerative disease, and accidental death from, you know, strengthening the exoskeleton. So that's clinically the question I'm most interested in as it pertains to exercise. But I'm convinced that at the center of that question is understanding the role of exercise in mitochondrial health. I think this is a very important piece of the puzzle and certainly much more important than I appreciated even two years ago. I think what you describe about these age appropriate or age acknowledged declines in VO2 max mitochondrial density, mitochondrial efficiency, venous O2 concentration, I think there's something really important there. And even if exercise is affecting something upstream that is affecting that, at least we have a great proxy through which to measure.

I think there's going to end up being a lot of really interesting nonlinear dynamics of mitochondria as a function of age, as a function of exercise. There's a few vignette cells share. As you probably know, if you don't use your skeletal muscle, you lose lean muscle mass, you lose your mitochondria very quickly. How quickly you have measurable defects in the VO2 Max after 10 days of hospitalized bed rest. And to recover the VO2 Max that you lose in 10 days, it takes about six weeks or so.

Oh, my God. Yeah, I knew it was bad. I didn't know it was that bad.

So there's quite a bit of hysteresis over here. Quite a bit of hysteresis. So it's going to be complex and nonlinear. The programs that turn on mitochondria during exercise, they're really elaborate. And the idea of actually replacing it in a pill may end up being kind of naive. I think that exercise does so many things simultaneously. It's like 17 different inputs into the system. And it may be the case that it's only if those 17 inputs are provided with the right dynamics and the right off rates that you get properly functioning more mitochondria in certain disease states. Some of the muscle disorders that I study, the ragged red fiber that you may remember from your board exams, the ragged red fiber represents an accumulation of poorly functioning mitochondria. So I think that if you try to bottle up just two of these factors or three of these factors, we may be able to produce more malfunctioning mitochondria. But it could be the case that we've evolved to require 17 inputs provided at the right time and place in order to get proper mitobiogenesis. It's a really, really smart program, this PTC1 Alpha program, because it simultaneously turns on mitochondrial biogenesis while also turning on some of the autophagy programs. And so you're actually turning over your bad mitochondria while you're turning on your good mitochondria simultaneously. And that's what happens with exercise.

Well, you read my mind, and I don't know if you could read my little notes I'm taking over here, because as we're talking, I'm making little notes of things I want to ask you. And that's exactly kind of where I wanted to go, which was, let's talk about what autophagy means in the context of mitochondria. So people who listen to this podcast know that I'm a big fan of fasting, periodic fasting. Because even though we don't have great ways to measure autophagy clinically, I think we have pretty good evidence that periods of really strict fasting, meaning exclusively consuming water for some period of time, my hypothesis is three to seven days, produces meaningful autophagy. But how does exercise impact that based on what you've seen in the mitochondria?

I don't know too much about fasting, but when you do have proper exercise regimes, what we observe is that there are transcriptional programs with multiple inputs, some of which we discussed earlier, but those are probably not sufficient, that will basically turn on all 1,000 of those proteins to produce more mitochondria. But that same program is also saying, hey, let's turn over some of the previously produced mitochondria. It's a very, very smart system. It's not going to just produce more good mitochondria in the presence of bad mitres. It'll actually cleanse the system as well.

And remind me what you said. I know you already answered this. I apologize. When you look at cells that are not turning over quickly, so myocytes, neurons, what did you say was the approximate turnover of mitochondria?

Probably a few days.

A few days. Unbelievable. So it's just. This is an unbelievable amount of work to create the new and systematically and selectively discard and recycle the old.

That's right. That's right. And the signals. Given that you like exercise. I'll tell you one study that I thought was really provocative and maybe you already know it, but it came from, I think, Michael Ristow and ron Kahn about 10 to 12 years ago in PNAS. Do you know this study?

I don't. I don't think I do, but keep going.

It's a human study.

Okay.

Two by two matrix. They randomized humans either to exercise or no exercise and antioxidants or no antioxidants.

Ah, yes. Okay, I know the study, but please keep going. No, this is great.

So which of the four quadrants do you think is best?

Well, I know the answer, so I want you to. Yeah, you keep going.

Yeah, yeah. So most people would probably guess.

Most people would say exercise with antioxidants must be the optimal health.

Absolutely. And what this study showed that was a little bit counterintuitive, is that antioxidants on top of exercise almost prevents or erases some of the beneficial effects of exercise. The authors concluded that things like reactive oxygen species are probably playing an important signaling role as well that helps in the adaptation you need some of those sparks in order to turn on new programs that are net beneficial. So if you erase those sparks, yet you prevent the full benefit of exercise. Just another reason why the entire system is so complicated, I think, investigating exercise. And again, we don't do that. That's not a core scientific focus of our laboratory. But so many diseases, ultimately their risk is reduced by exercise. So studying it should be a very important objective for all of us.

It's so interesting because that is a great example. I'm glad you brought that up. And Nav and I though we didn't talk about that study, we talked a lot about this issue of blocking ROS and how if one has cancer, for example, the evidence is becoming pretty clear that the last thing you want to do in a cancer patient is give them an antioxidant as sort of anti, sort of dogmatic as that would seem, because the ROS actually play an important role in selectively targeting a cancer cell versus a non cancer cell cell. Listening to these discussions makes you almost wonder, how in the world does any drug show up with a benefit in longevity? It's almost a miracle that rapamycin can so ubiquitously across so many species, extend life when as you point out, most of the things that do the heavy lifting and longevity have 17 prongs that can't be replicated by a single molecule. I mean, it just, it seems impossible. The one that has me very interested right now, and again, I don't know how much you've studied this. My guess is even if you haven't, just your peripheral knowledge will exceed that of anybody's is metformin. So again, I think most people listening to this podcast know a lot about it. I had an interview with Nir Barzilai, who I'm also having lunch with today. And Nir's certainly one of the world's experts on this topic. So we had a great discussion of all of the benefits of metformin. Don't think it's really disputable how big those benefits are in people who have diabetes. I think that is becoming very clear and then by extension in people who are insulin resistant. What I think is not entirely clear and I think is the purpose of what Nir is hoping to study with TAME is if you took a non diabetic, non insulin resistant individual and gave them metformin, will you enhance their longevity phenotype? And the one area that I'm most interested in this question is what is the impact of metformin on the ability of exercise to improve the phenotype. And that's something that just on a personal level, I've been experimenting with a lot. So doing a lot of lactate testing on myself with and without metformin, and using lactate as a proxy for mitochondrial function. So, you know, we were talking about this a little bit before, but just for the listeners of what I do is take a resting lactate level. I shouldn't be using any more ATP than I'm using at this moment. What is my level of lactate? Then on a bicycle, that allows me to control the wattage to the nearest watt, basically move in 5 or 10 watt increments slowly, you know, spend 10 minutes at this wattage, go up by 5 for 10 minutes, go up by 5 for 10 Minutes, and keep measuring lactate levels. And you generate a performance curve, an lpc, a lactate performance curve. And you do this with and without metformin. You see a difference. The question is, does that difference matter clinically? And is it possible that metformin is actually not helping in the context of exercise?

Are you saying that in the presence of metformin, if you are exercising, you're producing less lactate or more?

I'm seeing more lactate in the presence of metformin. Now, again, this is an N of one study on myself, but it makes sense that you could. I mean, that's a plausible.

How are you measuring your lactate using.

Blood in the finger?

Yeah, we should do metabolomics on you. I mean, with our new instrumentation, we can measure not just lactate, but literally measure everything. Hundreds of metabolites. It gives a little bit more of a comprehensive snapshot of all of your metabolic pathways.

Could we get an IRB to do that?

Easily. Let's do it.

I'm all in. So we could do it. We could do an on metformin off metformin snapshot, because so here's my crude thinking on this is if metformin is inhibiting complex one, it wouldn't be beyond the realm of possibility that the body might preferentially not shuttle pyruvate into the mitochondria. I mean, it's still doing so to a great extent. But if it's disproportionately now keeping pyruvate outside and turning it into lactate, that could drive up lactate levels. The thing that surprised me the most is how high my resting lactate levels seem to be. I mean, I remember before I started taking metformin, you would barely check a resting lactate level, but it was usually below 1 millimolar now, my resting lactate level on metformin is typically between 1 and 2 millimolar. It's about 2x. And I'm doing this in as painful and, but hopefully valid a way as one can do it, which is I'm using two separate meters checked in duplicate on the third drop of blood. Like, I'm trying to be as systematic as possible. Anyone listening to this? Who wants to do this? I just want to warn you in advance before you get started, lactate meters are upsettingly expensive, and the strips are the racket. I mean, they'll sell you the device for 300 bucks and each strip costs you about $5. So every time I do one of these dumb tests, which I typically do about once a week, it's like dinner in a movie for five people. But it'd be amazing to see what a broader sequence of metabolomics looks like to understand is there something that's happening. And by the way, then the next question is, do that in somebody who has diabetes and see if you see an improvement or a reduction in performance.

You measured your fasting resting lactate before you started metformin. Do you know if it went up after you started metformin and then it went back down again or.

Well, so that's an interesting question. So before I started taking metformin, I would do lactate testing, but I was interested in a different question. So it's not an apples to apples comparison. We always had a resting lactate just because you wanted to basically calibrate the machine and make sure your machine was working. But generally, when we did what was called this lpc, the lactate performance curve, it was mostly geared towards identifying a different position on the curve, which is very crudely done. But you do a series of efforts at different power outputs or in a swimming pool at different speeds or on a track, different running velocities. And you'll notice that the curve is nonlinear. So it starts like this. I'm sort of drawing, it goes flat, and then it starts to asymptote. It starts to shoot up very quickly towards a vertical asymptote that can be approximated by two linear curves. And the intersection of that curve is generally a person's lactate threshold. And that's different. That's usually a higher number than 2 millimolar. Let's just say, to make the math easy, that usually is in about the 4 millimolar range. And it's that point where that corresponds to on the x axis. That output is generally about the fastest Velocity or output a person can hold for a certain type of race that we're interested in studying. So I have infinite numbers of those data for myself back in the days long before I took metformin. But it was undeniable how low my lactate level started. So I at least have that data point.

I think that's really interesting. We published a paper in PNAS a couple of years ago where we placed either healthy individuals or patients with mitochondrial disease on a treadmill, did a 10 minute exercise test, and then drew their blood at rest, peak exercise and post recovery just to look at the metabolome response to exercise. So of course we get lactate in the mito patients, some of whom have complex I deficiency, not because of metformin, because of a genetic deficiency. They begin with a high resting lactate and there's a parallel rise in their lactate that parallels what happens to a healthy human and it stays high parallel with the healthy individual post recovery.

Do you remember offhand how high their resting lactates were relative to the non insulted?

It was single digit millimolar. It wasn't sky high. I want to say something like 2, 3, 4 millimolar, but we should look that up just to confirm. But it'd be interesting to see this is purely science. Now talking to you is whether we could repeat that exact same study not with genetic complex 1 deficiency, but with metformin.

Right, Exactly. Yeah.

You know, a lot of people ask me about metformin and aging, and again, we don't do aging, real aging research in our lab. We hope to be able to impact that through some of our work.

And I'm hoping that my questions are like prompting you to celebrate your. Yeah. You've got all these amazing tools to study it, right?

Absolutely. It really is a space that really captures anyone's imagination. But if you ask me how I think metformin is working, I think it's probably related to the body's homeostatic response to complex one inhibition. So of course metformin hits complex one. I think that's undeniable. It may have other targets, but without a doubt it hits complex one. When complex one has been blocked, the body senses it and there's a feedback loop, there's a homeostatic response, and that's probably what is net protective or helpful. And it may be the case that throwing a wrench in at complex one, it turns on 15 of those 17 inputs that you need to sort of rejuvenate not just your mitochondria, but other parts of your Cell as well. I think there's some really interesting experiments in worms. As you probably know, there's quite a bit of worm longevity work and there was early studies by my MGH colleague Gary Rufkin, as well as Cynthia Kenyon, who was at UCSF and is now at calico. They did RNAi screens to basically look for genes which, when disrupted, would lead to a longevity phenotype. One of the gene sets that was most associated with a longevity phenotype was the mitochondrial electron transport chain. At the same time, one of the gene sets that was associated with a drastically reduced lifespan was the mitochondrial electron transport chain. You can ask the question, it's a different subset of genes. Obviously there's about 90 genes total required for the electron transport chain and oxidative phosphorylation. So the question question is why do loss of some of them lead to longevity and why do loss of others lead to a shortened lifespan? One hypothesis is that it's just the strength of the allele. If you. Some of those RNAs really wiped out the electron transport chain, probably led to early death of the worm. But if you just gently block the electron transport chain with the right RNAi allele, perhaps mimicking what metformin does, you do get a longevity phenotype. So a hypothesis in the field, I'm not alone, but I think there's others in the field that think that maybe one of the ways that metformin works is sure it does block the electron transport chain, but then it comes back and causes an entire adaptive or a homeostatic response that is net adaptive at the whole organism level.

I'm not going to put you on the spot and ask you if you think people should take metformin. I think the broader question is do you think based on what you've seen in the ETC models that it's quite possible that a drug like metformin can be beneficial to some and harmful to others?

Oh, I think without a doubt. I mean, we know that metformin is useful for type 2 diabetes. So I think it's a fact that for a subset of the population, metformin is helpful and beneficial. In a rare subset of cases, you can actually have fatal lactic acidosis from drugs related to metformin. Things like fenformin.

Right. Which is more potent.

That's exactly right. That gets back to this idea of the potency or the allelic strength of inhibition of the electron transport chain.

But if you took that acute toxicity aside, I mean, I think this is really the question I've now become fixated on. If you take somebody who is already maximizing the benefits of exercise, nutrition, sleep, these things that I think the more we look at them, the more powerful they are. It's one thing to say the addition of metformin offers minimal benefit or incremental benefit. It would be another thing. If you're more in the Ross category that you alluded to earlier, is this actually undoing some of the benefit?

That's why I actually think that the experiment that you and I just discussed a few minutes ago, trying to see what the cross product of exercise and metformin look like, I think it could be totally fascinating. Is it gonna look like the Ron Kahn study from a decade ago where.

There is at least one experiment out there that suggests that. But again, I don't know how deep they looked at this. So this is a very, very interesting idea. The only drawback of this idea means I have to keep coming back to Boston. But now we're entering the right time of year to do it. So that's right. That's fine. Yeah, yeah. So changing gears around the role of hypoxia as a therapeutic, I mean, based on what you see in the mitochondria, how do you see that as a potential therapeutic option?

This is something that we're really excited about on the pre clinical level. And I really want to emphasize this, Peter, because oxygen follows the Goldilocks principle, right? I mean, too little is absolutely fatal, deadly. What we're discovering is that too much in certain instances, genetic backgrounds can be damaging as well. And so all of our work to date has been focused in preclinical models. One of the things that we are discovering in these rare mitochondrial disorders is that a lot of the ATP levels are actually nicely defended by glycolysis. And so although the textbook dogma is that a lot of these disorders are disorders of energy deficiency, under resting conditions, ATP levels are okay. But what we're observing is high unused oxygen. And important question is how can we now try to interdict and somehow try to reduce the delivery of oxygen? So at least in our mouse models, we're using hypoxia chambers. We actually dilute the air that the mice breathe with nitrogen. We use some of the devices that the sports industry has created. Nitrogen generators, face masks, tents. We place the mice in those apparatus, dilute the air with nitrogen, and then we evaluate the impact. And at least in some, not all, some of our mouse models of mito disease, the benefits are striking.

Let's explain again just why this is so profound. Right. So going back to the model you described earlier, in a subset of clinical scenarios where mitochondrial function, for lack of a better word, is impaired, you're seeing a much higher level of oxygen return to the lungs despite a high output of energy, imputing that their mitochondria simply aren't working. That high amount of oxygen itself can be problematic. So you're saying, well, rather than putting you in an environment where the ambient oxygen concentration is 21%, we're going to lower that. How much do you lower that to, by the way? In these tents with the mice at.

Sea level, we're typically breathing about 21% oxygen. We reduce it down to about 11%.

How is that? I don't know much about my altitudes, but that strikes me as really low. Is that like the top of Mount Everest low?

It's not Everest. It's probably base camp.

So it's 18,000ft. Everest is what, 7%? Maybe?

Exactly, exactly. So we're talking about certain parts of Bolivia. We're talking about Mont Blanc.

Yeah, yeah. But my brother's been to base camp, and he did something really funny, which is something only my brother would do. He typed out a series of questions for himself that at sea level, the answers to which are patently obvious. Ten questions. And at 10,000ft and 15,000ft, and then at base camp is 18. And then I think when he was there, they ended up not being able to cross the ice walls, but they could still get to, like, 21,000. And then again at 21,000ft, he would video himself answering these questions. And it was actually quite interesting. Not just the huffing and puffing that invariably goes into it, but the length of time it took him to think of the answers, which, when you consider the fact that he was answering the same questions and over and over again, it should have been the opposite. It should have been easier and easier and easier to come up with, what year did such and such happen? Or whatever. That's no joke, right? I mean, that's still, for many people, quite a deficit. So how did they improve?

Earlier in the conversation, we were talking about this disease called LI syndrome. So we have a mouse model of LI syndrome. It's actually due to a loss of one of the subunits of complex one. So it's a recessive loss of one of the nuclear subunits of a complex one. And this mouse is born looking okay. It's developmentally okay. But then right around day 30, 35, it starts looking sick. And by day 55, it'll basically fulfill our hospital's euthanasia criteria. It's lost body weight, it's become hypothermic, it's very, very sick. It has lesions on brain mri.

Is there anything in its periphery that looks lesioned or is all of the insult to the brain?

This initial cause of death is probably brain driven. And if you look carefully, other tissues are affected as well. So that's what happens. At 21% oxygen, there's a very stereotyped trajectory. These uniformly fatal at about day 55 or day 60. If these mice are grown at 11% instead of 21%, they now survive to about a median of one year.

Oh, my God. So you've restored them to half their normal lifespan.

That's right.

And what's their function level? How do they interact? Do they act like normal mice? Up until then, when we were doing.

These experiments, a very talented former graduate student, she's now at UCSF and our lab manager over at mgh. They're doing these experiments. They actually thought that there was a genotyping error because the mice looked so good. We actually thought that we had mis genotyped them. And so they look great. They put on body weight, they put on body temperature. The results are striking. But again, I want to emphasize that this is all an animal model still.

If you're a parent who one day has a child born with this condition, to think that the answer could be your child, instead of living a few months, could live into their 40s or 50s by moving to a part of the world, which would be the easiest way to accomplish this. I don't think it would make sense to live at sea level and wear an oxygen deprivation mask for your entire life. But if the answer is, guess what, you're going to go be a Sherpa, I mean, that's unbelievable to think of. I mean, that's. I would not have predicted that at all, based on what we've discussed, that it could be that strong in effect.

Well, what's interesting is in the past, people, that you proposed hyperbaric oxygen as a way of rejuvenating one's mitochondria.

That's what I would have stupidly suggested also, which is, wait a minute, we got to try harder to get that oxygen in there. Let's go hyperbaric. So have you done that in the mice? And did the mice die even faster under hyperbaric conditions?

So we didn't try hyperbaric because that's higher pressure, but we just tried hyperoxy.

Okay, okay.

So we went up to 55%, which is what is often given in the operating room as an example. The mice will die within a few days of exposure to 55% oxygen. So there's something about having.

And what about hyperbaric at 21%? Has that been tried?

We haven't tried that yet, but just hyperoxic at 55%. These mice will die within a few days of exposure. Peter, Within a few days of us publishing that paper, we actually got phone calls from across the country of cases where patients that were on the outpatient had been placed in hyperbaric chambers. And then they actually ended up, in some cases, dying within 24 hours or going blind in a good eye within 24 hours. And so I think there's some anecdotes that suggest that super high oxygen levels on a broken electron transport chain can be very damaging in humans as well.

Well, this is sort of interesting, right, because on the cancer front, people have talked about hyperbaric oxygen being a very potent tool because the mitochondria of cancer cells are going to be defective on balance relative to the non cancer cells. That's, again, outside of your wheelhouse. But how do you think about hyperbarics in terms of a tool to selectively target cancer mitochondria?

We're super excited about it, and in fact, we're really not a cancer biology lab. But there is a subset, a very, very rare subset of tumors where we're currently exploring that idea. I think others have thought about trying to starve cancers of their oxygen and glucose. Our idea is the exact opposite. Maybe in certain instances you want to flood them with oxygen.

Do you have a sense of which cancers in humans might be more or less susceptible to that pressure?

So we're looking into that now. But it's going to be a rare subset of cancers where there may be some mitochondrial mutations to begin with.

So in other words, it might be less about the given histology and more so. You know, it's funny, I talked with Keith Flaherty recently, and this is a great example of targeted therapy in cancer, right? Imagine you have your tumor, you get it sequenced, and you realize, oh, look, you have a tumor whose mitochondria are especially weak. You are a great candidate for hyperbaric oxygen. Person B. Over here, the mitochondria in your cancer cell look perfectly fine. Hyperbaric oxygen, if anything, is not going to do. At best, it's going to do nothing. At worse, it might actually harm your other cells.

We've looked a little bit into this literature. We have an oncologist in our laboratory that's looking in this direction. And hyperbaric oxygen has definitely been proposed as a cancer therapy in the past. But there have been mixed signals. And exactly as you're saying, it may be the case that if you know how to precisely target it, maybe you'll see a real signal.

Anything else in your work have you thinking about cancer? This is a great example. Is there anything else that you think about with respect to what you've learned and how it pertains to cancer prevention or treatment?

We had a series of projects about five, six years ago or so where some of the guys in the laboratory were looking at sort of omic data sets from large numbers of cancers, Just asking, what are the most consistently altered metabolic pathways ways in cancer? So there's about 1,500 metabolic enzymes encoded by the human genome. Which one is the most upregulated or down regulated across all cancers? And that pan cancer analysis, it wasn't a mitochondria focused analysis to be clear, but it revealed a few mitochondrial enzymes in the folate pathway that are the most consistently upregulated enzymes across all cancers. The mitochondrion is a powerhouse of the cell. It does produce ATP, but it's also a biosynthetic machine as well. There's a few pathways within the organelle that are designed to produce one carbon units for growth, folates, things like that. That pathway was highly, highly upregulated. It gave rise to the seductive idea that maybe mitochondria are not being used for energy in cancer, but rather as biosynthetic machines for cancer. So that was an idea that we and others stumbled upon about five, six years ago or so. But it's not an active area of research in our lab.

Well, it's interesting because it's very consistent with other hypotheses that the Warburg effect is less a deficit of the cancer cell due to defective mitochondria or inability to undergo oxfos. And maybe the Warburg effect is the result of a cancer cell wanting to get a higher throughput of substrate to foster growth. Obviously, Matt van der Heyden was the author on a paper that talked about that several Years ago, about 2009. Hadn't heard the folate story. So that's kind of yet another really interesting point. Single carbon biology is pretty interesting stuff.

That's right. That's right. And several investigators like, like Josh Rabinowitz, David Sabatini, our group.

I had dinner with David last night.

Great.

Yeah, we actually were talking about single carbon Metabolism and the challenges of it. I went to med school with Josh, by the way.

Oh, wow.

Yeah.

Yeah.

Oh, wow.

Okay. So actually the three of us, about five, six years ago or so, we all had sort of independently stumbled upon this mitochondrial pathway as being dramatically upregulated. Josh with metabolomics, David with RNAi, us with computation. And I think to this date, there's a lot of data that supports the idea that this is upregulated in cancers. Now, whether targeting it is going to be beneficial, that's an open question still, but without a doubt, this is one of the pathways that is powerfully upregulated in cancer states.

On the topic of cancer, and we've talked about these other chronic diseases, but it doesn't really appear that there is a chronic disease in which the mitochondria remain normal. If you look at cancer, if you look at Alzheimer's disease, if you look at atherosclerosis, and if you look at type 2 diabetes, all of these diseases have mitochondrial signatures that differ from what we would consider healthy.

Well, it gets back to the opening parts of the discussion where we said that if you take any aged tissue, it's going to be associated with dysfunctional mitochondria. And if you take diabetic muscle, if you take Alzheimer's brain, Parkinson's brain, you're going to see dysfunctional functional mitochondria. And this is why it gets back to, is it cause or effect, or is it going to be some complex nonlinear combination of cause and effect? And this is where using that systems biology approach or trying to gain insights from these rare diseases may inform a subset, but not all of these. I actually think that for Parkinson's, the causal hypothesis is pretty compelling. Out of all of these, these disorders.

Say more about that, Because I don't know anything about Parkinson's that I didn't learn in medical school, which, if I recall, is more to do with dopamine secretion out of a part of the brain that the substantia nigra. Is that it? Where you basically lose that neurons? Yeah. And so the patients that maybe start out with genetically fewer of those, because there's a distribution of how many you get, maybe the ones most susceptible. I never thought of that as a problem that shows up in the mitochondria. So expand on that.

Again, we have two classes of disorders, right? We have sort of common, complex diseases, and we have monogenic diseases. And the big question is, does the pathology we see in the rare monogenic forms bear any relevance? Right. To the common forms? Of disease.

It's funny, I mean, the way we're talking about it, your hands are showing it in a way that I think think is representative. Right. You had your one hand out over here and you said, look, these are the very simple monogenic diseases. Most people have never heard of them. They're typically quite brutal, but they kill relatively few people. On the other hand, you have. There's a divide, right?

That's right.

And it's like. It's quite discontinuous, isn't it?

Well, also on this side, the prevalence of these monogenic mitochondrial disorders is about 1 in 4,000. But then as you cross the continuum.

The prevalence is one.

One in one.

Yeah, exactly.

And then you have these other disorders like type 2 diabetes and Parkinson's and Alzheimer's, that it's not one in one, but it's also not one in 4,000.

So you're saying Parkinson's may be the closest example that you can think of that's spanning these two worlds?

I think so. I think so.

Okay, I want to hear more about this.

Well, I think there's a couple of reasons. One is if you take the common form of Parkinson's disease and if you take some of the post mortem material, if you biopsy that, or if you take postmortem material and you look, you'll actually see mitochondrial lesions. You'll see an increase in the mutation burden in the mitochondrial genome, you'll see complex one deficiency. Already we're seeing some of the molecular features of mitochondrial dysfunction. But perhaps even more compelling is that there are some toxin forms of Parkinson's. Certain types of herbicides and insecticides are actually toxic to complex one.

So you're saying there are people who have Parkinson's, and I apologize for my ignorance on this, where they don't actually have a dopamine deficiency in the brain, but they have a Parkinson's like phenotype purely from an insult to their mitochondria from, say, a toxin.

Right. So in these instances, what we think has happened is that because of a environmental pesticide or insecticide, the mitochondrion has been poisoned in some of these dopaminergic neurons and those neurons actually die. So there is a dopamine loss.

I see.

Okay. But the root cause is the mitochondria. That's right. That's right. So that's toxin evidence where we know that a direct toxin to the mitochondria in humans can give rise to a Parkinsonian like disease. In mouse models, if you give a high dose of Rotenone.

Is that a.

It's a complex one poison.

Okay.

And this actually gets back to metformin. Exactly. So if you infuse into a mouse rotenone, which is a very potent.

More potent than phenformin.

Yes. Right. And there's some potential off target effects.

As well, like micromolar or millimolar potency.

Micromolar potency. It may hit other things like microtubules as well, but it definitely hits complex one of the electron transport chain that's been used as a model of Parkinson's disease in rats and in mice. And so I think between the toxin evidence, the fact that sporadic forms of Parkinson's disease can be associated with complex 1 deficiency or mitochondrial mutations, I think helps to support the idea that mitochondrial dysfunction can play maybe in the causal path for Parkinson's disease.

Are you optimistic that. That we are going to be able to target mitochondrial proteins as therapies? I mean, the more I listen to this, the less optimistic I am, truthfully. Just because of the exceptions being exceptions.

And not rules, I'm actually pretty wildly optimistic.

Okay, that's great, because I'm coming away, like, discouraged. Like there are too many moving pieces to be able to use a single molecule. So are you thinking of stacking molecules or tell me where your optimism comes from.

Well, it comes from the fact that five years ago in some of these mouse models or cellular models, we had zero ways of alleviating mitochondrial disease in a dish or in a mouse. Now we have in our laboratory, we can use again in a preclinical way. We can use hypoxia and it actually helps helps to restore cellular function and longevity and health span. In mouse models of MITU disease, we're using other approaches that are evolutionarily inspired. We call these protein prostheses, where we take proteins from other organisms, from other kingdoms of life, we transplant them into human cells with mitochondrial disease, and we can effectively rescue the cells. And so how do you, how do.

You transfect all the cells? Or are you just saying that you can just. You're just, you're doing this ex vivo?

This is all ex vivo right now. Right.

But still, it's a proof of concept that's very powerful.

I mean, nowadays we have nucleic acid therapeutics, gene therapeutics, protein therapeutics. So.

So give me an example of one of the protein prosthetics.

Getting back to the earlier part of the conversation, we spoke about the fact that the electron transport chain was probably one of the earliest features of, of the early eukaryotes. Probably resembling the electron transfer chain of bacteria that can do oxidative phosphorylation. But then during reductive evolution, certain organisms lost parts of their electron transport chain. Now there are certain organisms that have lost their entire electron transport chain. And we think that one of the ways that they're able to survive is that they gained a new protein that basically complements part of the activity of the electron transport chain. We've identified some of those proteins. If we place those proteins in a human cell, you can poison the mitochondria any of five different ways and the cell will still proliferate because it has that protein that evolutionarily, we believe, allowed that organism to lose its etc to begin with. Does that make sense?

Yeah, it just seems too good to be true.

Well, this is why we call it a prosthesis. It's not 100% fix of the solution. What it does is it probably corrects part of the redox imbalance in the electron transport chain, but not the full proton pumping capabilities.

Yeah. So play this forward. You now could argue another treatment for type 2 diabetes is in addition to making the changes we're gonna make. Right? Because I still believe deep down that you can cure type 2 diabetes with corrective exercise and nutrition and sleep. I think if you get those things, three things fully optimized, type 2 diabetes goes away, with the exception of the late stage cases where the pancreas no longer works. But what if you could add another layer to that which is, oh, by the way, here's some mitochondrial prostheses.

That's exactly right. It's not going to fix all of the functions, electron transportation, but it can.

Become additive to other things.

That's right. That's right. That's one of the ideas that we're exploring right now. Can we actually, in the context of things like fatty liver and diabetes, can we use some of these mitochondrial protein prostheses to either augment or bypass some of the broken functions of mitochondria to restore function?

I don't know if you've ever thought of this because the idea just popped into my head when you said mitochondrial protein prostheses. Have you ever thought just in sort of random sci fi like thinking of the opposite, which is performance enhancing prosthesis for mitochondria. I mean, if you think about these, the efforts that athletes can go to to enhance aerobic performance. The most obvious of these, of course, is blood doping and use of epo, which simply deliver more oxygen to the system. But in 30 years, will people Be talking about genetic cheating where mitochondrial performance and function has been enhanced.

Well, it's funny because the sports industry is so often, I mean, the sort of underground illicit sports world of doping often is so ahead of the medical world. It's actually pretty amazing.

Yeah, it really is.

I mean, one of the compounds, it's like EPO, it's called FG 4592. It's a small molecule that blocks one of the prolyl hydroxylases. Basically tricks the system into thinking that it's hypoxic and you end up producing more epo. That drug was in clinical trials and before the drug was even approved, it was being used by cyclists, which is already amazing. But what's also even more amazing is that the Anti Doping Agency knew to look for drugs that were not yet even approved by the FDA in some of these athletes. And so it's like this red queen where everyone has to stay ahead of everybody else.

Yeah, it's an arms race. Do you think, like, theoretically, there are ways to enhance performance through. Again, I'm not a huge advocate on genetic engineering. I still think it's so. There's so much sci fi and the limitations of actually getting a virus that could be taken up ubiquitously. But putting that aside for a moment, could you engineer a better mitochondria? Could one? I mean, I don't want to put you on the spot, but could one engineer an even better mitochondria? How much waste is in the system? It's funny, if I ever did this exercise, it's so long ago that I don't recall, but from an engineering perspective, how much room for improvement is there?

I think it's going to take a while before things like that are possible. And I gather from this conversation that you, like me, like automobiles. And so if you have an engine, the way to enhance it is to turbocharge it. But turbocharging it is not taking one spark plug out and replacing it with no, it's another spark plug plug.

It's completely changing the way air flows through it and is recycled.

So you have like 17 different things. You have a turbocharger, you put it on top, but there's like 17 other things that you have to alter so the entire system connects up. You have impedance matching, you have airflow matching so that in the end you have a better performing automobile. I think something analogous applies to mitochondria. It's not going to be just one thing that you can turn on if you want to try to. The system is already pretty optimized. Right. And so if you just change one thing, it's probably not going to. You can break it.

Yeah, you can break it with one thing, but it's hard to enhance with one thing. That's actually a great way to just think about biology in general. It's pretty easy to break it at a single point. Cyanide, I think the most extreme example. Right. Detrodotoxin. So easy to kill and break at a single molecule. Which comes back to a point I made earlier. It's mind boggling when something like rapamycin works.

Right. Again, I mean, you had dinner with David last night, so he's clearly one of the world's authorities on rapamycin and its function. But at least if we go to metformin, the only way that I can conceive that it could have this total body impact is if it's doing something that then causes the entire system to respond. Even when we talk about stat statins, every medical student in the country knows that statins are life saving. Every single medical student in the country knows that statins hit HMG CoA reductase. But why is that enough?

Right. Is there other things that it's doing that are also important beyond the reduction of ldl?

The way that statins work? My understanding is that sure, statins directly target HMG CoA reductase, but then what ends up happening is because you're not producing so much sterols, you turn on an entire SREBP transcriptional program.

Right. And you bring more receptors to the surface of the liver. That's right. More of the statin efficacy is due to the LDL clearance than the reduction of cholesterol synthesis.

That's exactly right.

But there may even be benefits beyond that is my point. And some argue there are actually a whole camp of LDL denialists who can't deny the efficacy of the drug. And so the sort of hypothesis is, well, all of the statin benefit doesn't come through the LDL reduction through the mechanism you describe. The unintended consequence is the wrong way of saying it, but the non obvious consequence that it could be some of the anti inflammatory benefits that come from it or the endothelial protective benefits.

But the point is that there's an entire response to hitting HMG COA reductase and a consequence of that program is that you have more sterile production. So in the end, the amount of cholesterol that you're actually producing is kind of balanced. You've inhibited it, but you've turned on more of the enzymes. So it's comparable, but you've turned on these other 17 switches as well at the same time, one of which is the LDL receptor, which helps to clear LDL levels. There may be other things as well that are turned on that are net beneficial. So I think physiological systems are so complicated. Trying to identify all 17 of those things and turning them on at the same time in general, I think is going to be hard even in the next 30 years. But it may be the case that some of the interventions are, you know, what people refuse.

Do you think of this as when you like. Let's look at the three examples you've just used. A statin, metformin and rapamycin. None of those are purely, I mean they're synthesized today, but they are all derivatives of naturally occurring compounds. I'm sitting here as you're telling this story, trying to think of examples of purely synthesized de novo molecules that have such benefit. And there may be examples, but it seems less obvious. Do you think it's a coincidence that some of the most potent agents that we have in medicine, which by definition it seems are the ones that have to do multi pronged inputs, tend to come from naturally occurring substances? Does that just speak to our co evolution with these things?

No, absolutely. I think there's been this arms race, right? The bacteria are fighting these fungi and the fungi are fighting these plants. And so there's all sorts of small molecules that are designed to throw wrenches into other mitochondria or translational programs or nutrient sensing programs. So natural products are remarkable chemistries and they've evolved over hundreds of millions of years to target physiological systems. And so I'm a huge fan of natural products and the biology that they expose.

This is so interesting. We'll close with a question that's admittedly kind of a tough question. So I apologize for putting you on the spot. And no, no qualms if you can't come up with a, a good or great answer. But when you think about the world you want to explore here with the questions that you want to ask, so much of what happens, even at the level that you're at, is still constrained by resources. Have you ever had the thought experiment or the sort of fantasy of what if you were in a totally resource unconstrained world, so you never had to apply for another grant, in fact you were given some lump sum of money that was beyond what you could imagine. And even just from an IRB standpoint, there was nothing that stood in your way of doing kind of something that was still ethical, but maybe today would be logistically too challenging. Do you have a sense of what questions you would want to probe, but specifically what experiment or set of experiments you would do in this dream state?

Yeah, it's a tough question. It sounds like you're basically asking me not to be limited by anything except my own imagination. And I think so often in biomedical research that is what limits us. But it's a great question, Peter. One experiment that would be kind of a fun experiment to try is really motivated by a recent work on oxygen. What we've observed is that at least in preclinical models, when you have severe mitochondrial decline, breathing thinner air appears to be beneficial. Now, how about in the common form of aging, when there's a subtle decline in mitochondrial activity, is there excess unused oxygen? And will breathing thinner air be beneficial? And given that I'm not resource limited, wouldn't it be cool if we could construct a Ritz Carlton hotel or condominium at 16,000ft that's extremely comfortable and perhaps another Ritz Carlton that looks identical at the plains? And we could take a very large cohort, not five, not 10, but thousands of people that live at either the plains or at the high altitude for many, many months. And we try to evaluate whether there are any biomarkers of aging, age associated disorders that actually improve under thin air. Maybe they become worse under thin air, but it would be something like a crossover experiment that would allow us to test the idea that thin air may be beneficial for chronic diseases.

I love that idea. That is elegant because one, you could do that. You answer the question right, which is in a resource unconstrained world, tens of millions of dollars would allow you to do that, call it hundreds of millions of dollars could do that longitudinally. And your intervention is elegant in that it's going to touch lots of things. That's interesting. So 16,000ft. So following up on that, have you done or contemplated doing the less beautiful version of that in mice where. Or I'm sort of disdainful of mice, but maybe rats or something that's less inbred, where you have models of type 2 diabetes but they're genetically born with normal mitochondria. Has that experiment been done?

We're doing those types of experiments now in of course, our focus is on some of these rare genetic mitochondrial diseases, but we're going into some of these other conditions, more common conditions that are also associated with mitochondrial dysfunction. So those are currently ongoing. When we get back to that dream experiment, what is interesting, Peter, is Experiments like that have actually taken place in humans. And they can be right.

They're natural experiments.

And one of the experiments, there's this one paper from the early 1970s that was published by the Indian army. It was the Indian army reporting on the health outcomes of a huge number of their troops who India has historically had border disputes with China. I think it was in the 1960s, India deployed more than 100,000 troops on the Indochina border. And don't quote me on the numbers, but about 25,000 of these people were at extremely high altitude and another 100,000 or so were at the plains. They were there for about five to seven days, years or so. And of course the food that they ate, the temperature, the activity, all of these were different between low and high altitudes. But one of the variables that was different was oxygen. And after I think it was seven years, the Indian army actually wrote this paper. It's only been cited a few, like maybe 20 times or so. They reported the long term health consequences and what they showed is that death from things like infections were much higher acutely upon going to high altitude. But if you look at chronic diseases like incidence of diabetes, stroke, cardiovascular disease, they were much reduced in the high.

Altitude arm even after they returned to the plains, I think. Or only as long as they stayed at the altitude.

Only as long as they stayed in the altitude. I don't think they had long term follow up actually. But it's this type of data that actually makes me wonder whether what we're observing in some of these rare forms of mito dysfunction and the interaction of mitochondria with oxygen, perhaps some of it could bear relevance to more common conditions as well.

Super interesting. I can keep going on, but you've been generous with your time, especially to a total stranger. This is really helpful. I want to thank you for the work you're doing and I want to thank you for taking the time to explain it to me and to a few people listening.

Thank you so much. This has been a lot of fun for me.

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