Welcome to the Empirical Cycling Podcast. I'm your host, Kolie Moore, joined as always by our co-host, Kyle Helson, and I want to thank everybody for listening, and especially if you're new here and you like what you're hearing, please consider subscribing to the podcast. And if you are a returning listener and you are, well, you're coming back because you like it, thanks so much for coming back. And if you want to support us because you're apparently liking it so much, a five-star review wherever you listen to podcasts always goes a long way. We've got a ton of those, and we could always use a couple more. A glowing review would always help as well. Thank you so much for all of those. And if you want to donate to the show because we are completely and always will be ad-free, you can do so at empiricalcycling.com slash donate. And if you want to join our, what I joke is our ultimate Patreon, which is hiring us for coaching or consultations, you can always email me at empiricalcycling at gmail.com or head over to the website and contact info is up in there. So, oh yeah, also Weekend AMA is up in the Instagram at empiricalcycling. I've got a bunch of questions to answer when we're done recording this podcast because we're in the middle of that as usual. But I didn't ask any questions for the podcast, which I often do. But I think today because, well, I told Kyle this ahead of time, we're going to end this episode a little bit of a downer. Because we've talked about all these mechanisms with hypoxia inducible factor. And we talked about the kind of potential applications. When's it active? When's it not active? How does it work? And we've looked at... We've looked at a study where it was knocked out. We looked at phenotype and performance. But before that, we also looked at a study on high-intensity training. And I think the title was something like, hypoxia-inducible factor dominates the skeletal muscle signature for early endurance adaptation or something like that. Go to Wastock 51 or 50, something like that. And it'll be... in there. So that's where we have been kind of going is like, what are the performance implications? But today, I think what we're going to do is we're going to actually pull back a little bit. We've got a cool study that looks at moderately trained people and highly trained endurance athletes. And we're going to talk about kind of the implications about this and use it to paint a bigger picture of stress and adaptation. I also want to start with a historical perspective because I was reading a paper from one of the authors of the knockout paper that we dug into in the last episode. And I think it seems like hypoxia or inducible factor has lost its luster a little bit. Like I was reading a paper from the mid-2000s that was discussing what was known about a hypoxia inducible factor. blah, blah, blah. It's activated in response to the drop in PO2, targeted genes of glycolysis, building capillary density, all the usual stuff. But it was discussing all of that in the same breath as what we now know are much more critical factors for muscular endurance capacity. So we was talking about... MPK, talking about our old friend PGC-1α, but it didn't give PGC-1α the weight that we give it now 20 years on. Kyle, have you seen this in the literature before? Do you ever read something and you think, wow, that's funny that they didn't know at the time that this was so important, or they thought this was important at the time, and now we know it's not? Yeah, I think that happens a lot of times. Physics and Astronomy. And people can get very excited and very interested in a certain measurement or a certain thing, and that's because you start to see evidence of something. And then, you know, 20 years later, they've done enough follow-ups where they realize, oh, this is not actually something. I would say that's probably more the case in astronomy and physics because it's, you know, you get this one universe and sometimes the universe can play tricks on you, basically. And so... But I would say it is the sort of thing where people, you know, scientists are people and they're not immune to the idea that something could be trendy or something is appealing because that's the new hip thing. You know, I think that there are trends and, you know, peaks and troughs in the popularity of lots of different topics or lots of different... lines of inquiry throughout every scientific field, and it just kind of depends, and then sometimes it makes sense, and sometimes it's like, oh, that was weird. You get, like right now, honestly, the big trend in astronomy and physics and stuff, a lot of things are either quantum, anything with the word quantum. which is an axe to grind to have so I'm not going to go into that and then anything related to like habitable worlds like looking for planets that are maybe like Earth that are somewhere else in the galaxy so yes well luckily the world of cycling endurance training is not at all susceptible to things like that he says dripping with sarcasm yeah yeah so I actually think that since I was reading that mid-aughts paper, it seems to me that besides the one that we looked at in the first hypoxia-reducible factor episode, that more recent papers have actually, and the research world in general has kind of forgotten about HAF's skeletal muscle effects, and possibly with good reason, which we'll go into in a little bit. But yeah, I was reading another paper recently also on the mitochondrial response to exercise, and they noticed that the electron transport complex 4 was downregulated, but they never mentioned hypoxia or inducible factor. They never went, oh, by the way, we probably know why this happens, it's because of this. They just kind of went, oh, that's odd. And so, like a nerd, I emailed the author and was like, hey, have you checked out this? And I sent them like eight references. and he was like, oh cool, thanks. So thankfully I wasn't like, yeah, I know, go away. But anyway, it also could be recency bias on my part because I've been obviously digging into the literature on this. And yeah, so I think by now everyone's mostly figured out whether consciously or not that the evidence points to hypoxia reducible factor not exactly being the best explanation for All Long-Term Muscular Endurance Adaptations. Because, you know, we've got AMPK, we've got PGC-1α, we've got sirtuins, we've got MAP-K, we've got Take Your Pick, the list is pretty long. And so, I think it's at least still important to consider the HIF's role itself, maybe not in terms of It's going to be earth-shattering in terms of determining how do we train, right? Because we try to do a little bit of that with these episodes, like what are the actual practical applications? I've said this before on the podcast, but I think it's been a while. It's hard to really bridge a gap between the molecular mechanistic research and actual real-world application. There are very few times when we can really go, okay, yeah, this is exactly... how we should train. And I think actually the last episode on phenotype was actually really informative on this because, Kyle, I remember when we were going through it, I was like, so what do you think happened with this? They've got this endurance phenotype out of the box because of this knockout, so is their performance going to be any better or worse? You reacted the same way I did when I was first reading it, which is that the endurance performance was basically identical, so phenotype did not necessarily yield a performance advantage. So there's a complicated relationship there. I wish more people had listened to that episode. It's really poorly performing compared to a lot of others that we've done, but I totally understand not everybody's into the super nerd stuff. Niche audience here, so we love having everybody who's listening right now and is going to listen right to the end really excitedly. So, yeah. We also don't have a lot of literature on the effects of hypoxia-inducible factor in trained athletes. We're going to hypothesize a little bit at the end, and we're going to try to get a 10,000-foot view on what evidence we do and actually do not have in terms of all of this stuff. So do you want anything else to add before we kind of go into a quick refresher on hypoxia-inducible factor? Yeah, I kind of wanted to, with so many things within physiology and science generally, There are so many questions that people still have, and, you know, what was the, I forget who was it that said in, like, the 1800s that, oh, like, we've probably, you know, discovered all the science that we're ever going to discover. And so that's kind of never the case, and so things, especially if they fall out of favor, like, people may not. Revisit these questions for a long, long time, and then all of a sudden maybe they'll become popular again. But there are always these different questions you can ask, and there's never enough funding and people to go around and actually answer all these questions all at the same time. And then additionally, sometimes measurements become easier over time where, say, someone has a question. 30 or 40 years later, maybe a new technology has been developed that makes answering this question easier. So people might then go back and say, hey, this technique they didn't have in the 70s now makes this so much easier. And so now we're going to go back and think about this. Whereas maybe in the 70s, it was kind of a dead end because you only had XYZ available at your disposal. Yeah. And if you knew how gene sequencing worked in the 70s, whew, Right, yeah, yeah, exactly. Or like, or just even all the things like before they had like green fluorescent protein, right? Now everyone's like, oh, we'll just make it, make that shit green, you know, and make it like easier to track and stuff like that. Whereas before, before you could do that, you're like, uh. Yeah, I actually remember 10 years ago, I would not work, or I knew researchers who would not work with any protein that you couldn't histag, which is where you just put a string of histidines. onto the protein. And then it's really easy to purify when you elude it through a column. Since like a bunch of histidines has a high affinity for, oh God, I think it was nickel. So yeah, anyway, it was something like that. And so it was easy to purify and they were like, yeah, I don't want to do any kind of work with blah, blah, blah. And actually one of the experiments I did was on... Cell Membrane Protein, and I remember purifying cell membranes, and they were the world's biggest pain in the ass to work with because you've basically got like lipids in an aqueous solution and they clump together. It's like, anyway, so there are a bunch of things that I think, yeah, you're right. With more techniques, you know, things would be more elucidated like on old questions. as they kind of, people go back into the literature and go, oh yeah, we never figured this out, did we? Maybe it's interesting. So I think here, like there is actually a, we do easily have the technology now to elucidate a lot of our questions, but I think the, as we'll see, it may not be, at least in skeletal muscle, I don't think that we're gonna have a billion amazing answers. So anyway. Or maybe it's not going to yield the effects that we wish it would. And we'll talk about effects in a minute. But anyway, quick refresher. Hypoxia-inducible factor in skeletal muscle responds to low oxygen tension. So that's low partial pressure of oxygen. The presence of oxygen actually tags half of hypoxia-inducible factor. It's got HIF-1α and HIF-1β. So alpha gets tagged for degradation, but beta gets made... Institutively. And so when O2 levels drop, the alpha and beta quickly dimerize. They smash themselves together, and then they go transcribe a bunch of genes. So that's their mechanism of action. And we'll talk about their degradation more in a minute. But previously in untrained men, we saw that like nine high-intensity... Interval Training Sessions over three weeks. There was a large increase of HIF-related transcriptional pathways over the entire study. That was a couple of Wattstock episodes ago. So we saw an improvement in or a giant jump in expression of glycolysis, glucose transport, iron transport, capillary formation, so VEGF, so vascular endothelial growth factor. There's more in terms of like nitric oxide signaling and all that kind of stuff. So that happened, but pretty quickly... We saw that the signal changed over the course of those nine sessions where things like VEGF stayed high, but I think it was the expression of glycolysis genes actually decreased and kind of went closer back to baseline, if not to baseline. Go check that episode out for exact details. So one of the reasons that the glycolysis thing happens is because you need the enzymes to break down Glucose, because in the condition of we don't have enough oxygen, what is your pathway for metabolism to generate energy that doesn't rely on oxygen is glycolysis. So that's the big kind of reasoning that we're getting both, hey, eventually let's bring in more oxygen with endothelial growth factor, but also in the meantime, we need to break the glass and get the fire extinguisher out because we're having a problem right now. Yeah. So, yeah. Oh, yeah, sorry. You could imagine that, you know, a long-term lack of oxygen might be bad. Yes. Actually, you are one step ahead of me because we're about to get to that. So, leading up to that, last episode, So thanks again to all three of you who listened to it. We looked at a paper that showed that there's a disconnect between phenotype and performance. So mice with HIF-1α knocked out of their skeletal muscle had a phenotype, so they had RER, fiber type, capillary density, mitochondrial markers, et cetera, that looked like that of trained wild type mice, but they didn't have the performance. So they were pre-phenotypically trained, but they weren't actually trained when you actually asked them to run. And also, I have this in my notes here. I don't remember when I wrote this or why, but I said it was not mentioned in previous podcasts, but there's worse performance in higher intensity exercises due to the hit that glycolysis takes without hypoxia-inducible factor in those mice. So I must have wanted to mention that at some point. So there you go. So our typical assumption, which is, let's call it having a phenotype target. Right? Like, oh, we need to do something that's going to increase our amount of, like, Type II fibers or Type I fibers or whatever in order to get this adaptation. It seems to be necessary but not sufficient for actual endurance performance. And nothing in that paper was measured that actually co-varied with performance improvements. And so we can really only speculate why that happened. Like, was it the location of mitochondria? Was it glycogen stores? We don't know. And we could speculate all we want, but we're not going to get anywhere. But I think that for something like that, like having a pre... pre-trained phenotype without the trained performance, like kind of figuring out what is happening there, I think would be a super, super interesting research question in the future. So I hope somebody takes that one up, if nothing else. Email me if you want to do that one. I have plenty of ideas for how to set that up. So we also discussed the Pasteur effect. and this is named for Louis Pasteur. You may or may not have heard of him. Saying that name out loud may be outlawed in the US soon. We don't know yet. And so this is where cells shift to more reliance on anaerobic metabolism when the oxygen supply becomes scarce, right? So like they were looking at cells in culture and knocking out HIF, stunted their cell growth in anaerobic conditions because if they can't sense the drop in O2, and shift their metabolism accordingly, and these are single cells, by the way, then they're not going to be able to grow. So it's sort of like having eyes or not in terms of just being able to see something. Imagine if you were a cell and you didn't have any eyes. Okay, no cell has eyes. So imagine if you were a cell with eyes. and you're like, okay, I need to like get through this maze. If you've got eyes, you can blaze through it, right? You can negotiate that stressor. If you don't have eyes, like you're gonna be wandering around, like maybe eventually you'll find your way out, but like for a while, it's gonna be real tough and probably you're not even gonna survive. So that's kind of the difference it makes to be able to sense a stressor or not. It's probably a bad example, but sorry, it's all I got off the cuff. People, on the other hand, we have eyes, but we are not single-celled organisms. So we obligately have to do both aerobic and anaerobic metabolism because we're not going to survive much at all without oxygen. Imagine if we could actually shift our entire body's metabolism to glycolysis. Wouldn't that be cool? Yes, but also no. But in those cells and culture, and also in untrained people, we also saw a small but measurable blunting of the expression of certain aerobic proteins. Some of the typical ones that are usually measured are straight synthase and Electron Transport Chain Complex 4, for instance. So the overarching point, and I think what I really want to illustrate as a more general principle, is that the eventual adaptations from hypoxia reducible factor alleviate the conditions that trigger its activation and the subsequent response, which I think we can generalize to any stressor adaptation feedback loop. Does that make sense, Kyle? I hope it does. Yes, I think that it's, you know, you can kind of think of, this is maybe like, you know, kind of a, that could be a general model that you could use for lots of things, like some sort of feedback loop where, you know, if you can't, you can't, if you can't measure it or can't, you know, sense it, you don't know, but your body and, you know, lots of other systems, you think of like, you know, robotics or autonomous control of things, you know, they all have are all really need and work best with some sort of, you know, both positive and negative feedback type mechanisms to make sure that your body, for example, wants to maintain some sort of homeostasis. But other processes, you know, industrial processes, whatever, might, you know, also want those things. Yeah. Yeah. And so when we're thinking about cells, whether it's us or single-celled organisms in culture, When we think about short-term adaptations, hypoxia-inducible factor activation is going to either very much reduce reliance on aerobic metabolism, like in cells and culture, or it'll slightly do it for us. And it's measurable, but we have not really seen an effect on performance. That's another experiment that could be done. I'm sure it's going to be very large and expensive, but... It could be done, I think. So I think that in the longer term, you know, this is more where our audience is going to be concerned. Like, okay, we're past our noob gains, we're past the initial like four, eight weeks of a usual study, what now? So like we saw in the first episode in Pretty Untrained People, like we said, the whole gamut of adaptations didn't actually remain forever. It's not like every single high-intensity training session that they got the same exact adaptations at the same magnitude. All that signaling actually changed the nature of it. So some of it moved more back to baseline. Some of it stayed very high, like VEGF signaling. But as we know, we can't grow capillaries forever, right? There's a big mass of capillaries. Yeah, like, where are you going to fit a muscle fiber in there? It's all capillaries. Anyway, so how does the response change in well-trained people is a big question. And so we have some evidence for this, but we don't have a ton. And so let's go over the one study that I found that really actually looks at this. two or three weeks. I did another search, and I used like eight different search engines, everyone I could find to look for a paper on this, and I never found one. So it either doesn't exist or my Google Foo is really weak. And it's probably more of the former, but I know my Google Foo, Kyle, is not as strong as yours, for instance. So we're going to just leave that out there. Anyway, so this paper is called Negative Regulation of HIF in Skeletal Muscle. of Elite Endurance Athletes, A Tentative Mechanism Promoting Oxidative Metabolism. Okay, so it doesn't give up the whole thing there, but you kind of get the gist of it. So the author is actually- It's tentative in the title. Tentative, yeah. So you can tell they're hedging a little bit. Yes, yeah. We are under no such obligation to hedge as much, but we will still hedge quite a bit. So the author started by reviewing negative regulators of HIF. So these are things that decrease its activity or its effects. And they speculated that trained athletes would have increased expression of all these negative regulators. And so here are the ones they looked at. Prolial hydroxylase. So prolial residues, so that's a proline, it's just an amino acid. On the HIF-1α subunit, these are the ones that get tagged for degradation under aerobic conditions. And so hypoxic conditions block this and allows the protein to not be degraded and to actually go make adaptations. So another one is factor inhibiting HIF, F-I-H, which just turns down HIF transcription itself. I love nested acronyms. I know, right? I mean, PGC-1α is like the king of nested acronyms. Sirtuin-6. is an NAD-dependent histone deacetylase, which basically means it's an epigenetic co-repressor of HIF-1. So a histone is the stuff that DNA is wrapped around, and acetylation and methylation will have effects on how easy it is for transcription factors like hypoxia inducible factor to actually get in there and get at their target genes. So that's the... the epigenetic regulation aspect of it. And the authors also note, kind of like we have several times, that the long-term endurance training seems to be the opposite of short-term effects of HIF on skeletal muscle. And so that's basically what led to their hypothesis that training activates HIF's negative regulators. And so we've been more focused on adaptations. alleviating hypoxia conditions, but it appears that the regulation happens as well so that HAF doesn't get too twitchy. You got to be careful around HAF. You don't want to let it run loose in your cells too much. You might actually end up turning your cells fully into anaerobic bacteria in culture or something. That'd be wild. Brewer's yeast. Yeah, which I think is the organism that Pasteur saw the effect in, if I'm not mistaken. Right. Yeah. Yeah, I think it is. Oh, very clever, Kyle. Yeah, you got that. Okay, deep cut. Okay, deep cut. All right, so in WASDOC51, we did see a fairly rapid decrease in some of the HAF target genes over the course of a couple bouts of high-intensity training, like we said. But this study, they did two experiments. They did a cross-sectional study. and did a longitudinal study. So in the cross-sectional, they compared moderately active people with the elite athletes because they're like, what are these people's baselines? How are they different? And the longitudinal study, they compared before and after six weeks of training in moderately trained men because they want to see how quickly do these adaptations arise? Like what is the approximate timeline of effect given this type of training? So in the cross-sectional study, they had 12 cyclists and triathletes versus 9 moderately active men. Unusual that you could recruit more athletes than moderately active people, but I'm happy for them. So the comparison between the groups, VO2 peak was average of 75 in the train group, which is higher than usual, I would say. for a study. Usually it's like 50 to 60. Yeah. Versus the average of 47 for the moderately active men. And the standard deviations were tight. They were around two. Usually it's like five to eight, maybe 10 if you got a really unhomogeneous group, but like this is super, super tight. Citrate synthase activity for the trained men was 0.56 versus 0.22 for the moderately trained men. And that's microcatals per gram of dry muscle. And I don't know if we've ever talked about a catal before, but it's the SI unit for catalytic activity, which is one mole per second. So in this case, 0.56 micromoles of citrate generation per dry gram of muscle tissue, which if you want to extrapolate that out to an entire quad would be a lot. I love thinking about scaling like that. Anyway. What we're actually after is looking at if there's more red tape, so to speak, like crossing guards, the hall monitors, whatever you want to call it, stuff that is interrupting HAF's mechanism of adaptation or its gene expression. So proleohydroxylases, PHDs 1, 2, and 3. These are the ones that I said, tag HIF for degradation during normoxic conditions. So what were the differences they saw? Well, they published a picture of a representative Western blot, and we see a ton more PhD II in endurance athletes. I mean, a ton. So it's a literal blob, so we're just kind of going by arbitrary area units. It's 3.4. units for endurance athletes versus 1.3 units for the moderately active men. Yeah, so that's 2.6 times more PHD2, but PHD1 and 3 did not show any significant difference really. They looked at some mRNAs as well, but we'll talk about that more in a second because I think it's also going to be illustrative of another larger picture thing. FIH, so here's our nested acronym, protein levels average 0.7 arbitrary units in the trained and less than 0.2 arbitrary units in the untrained. So that's about 3.5 times more. That's a pretty big difference, yeah. Yeah. And sirtuin 6 protein levels also in the trained people, five times more. And so what's our answer to our question? Unequivocally, yes, there's a ton more of the negative regulation of HIF in the trained endurance crowd. And we can't really be sure of the exact magnitude. Like if we had both groups do a four-by-five-minute VO2 max workout, what would the signaling look like? Like how different would it be? We cannot say they did not actually do that study. That would be another easy one to do. I'm just lobbing Hail Marys out there on this podcast, I guess. Hey, call me. Or don't call me. I'll be second author. It's fine. You just gotta... Oh, I don't even need author credit. I just want to see it done. I just want to know. Somebody just go do it, please. So that's it. That's the cross-sectional study. That's the long and short of it. pretty simple. So it doesn't take much to answer an easy question like that, just a couple biopsies. So in the six-week study, they had 24 moderately active young men with a VO2 peak of 48 do four 45-minute sessions per week at 70% of their VO2 max from their pre-trained VO2 max. They did not continuously. increase. They just said, go do this for a couple weeks, four times a week, 45 minutes at this pace, come back and see us again. And so what they saw was our PhD II levels, which we saw from previously were 2.6 times higher. They saw these go up to 1.6 times higher. They went from 1.5 to 2.5 arbitrary units. But they didn't really see a significant difference with PHD1 or PHD3. And they also didn't see a significant difference with FIH or sirtuin-6 proteins. But we did see increases in their mRNAs. So the PHD2 mRNA actually only went from about 12 to 17 arbitrary units. But it was the only protein. that had a significant increase. The rest for FIH and CERT-6 and the other PDHs, or sorry, PhDs, these were all around 2.4 times the increase of the RNAs over the course of a couple weeks. So, sorry, six weeks. So, and this is, you know, moderately, we would charitably call this kind of like sweet spot tempo threshold type training. Like this is probably not gonna be over, threshold for a lot of these folks. But that's basically the training they did. And so it seems like what's happening is the groundwork for tamping down of HIF's activity is being laid, but it looks like it's going to take a lot longer at that intensity in order to actually fully get to the super highly trained type of negative regulation of HIF. So does that all... hopefully make some sense. Yeah, I think it's interesting because it wasn't a super long amount of time, but thankfully long enough for actual measurable differences to appear. And I think that it's maybe interesting because for folks thinking like, oh, six weeks, sometimes how much work, how much training, how much improvement can you have in six weeks? Six weeks is definitely a sufficient amount of time to start seeing changes in your muscles and measurably so. So this is, I think, also interesting for that where people say, oh, I'm out of shape, you know, oh, six weeks. Six weeks isn't that long, right? It's a month and a half. That's maybe one training block and a rest week and then starting up another training block again. It feels like that's not that long. Yeah, yeah, and it's really not. Yeah, but it is enough, and your body is responding to that. Yeah, and it's a lower intensity than we've seen in some other studies. It's not like VO2 max every day for over six weeks. Well, we've looked at studies where that's pretty much been done. Yeah, no, I know. Yeah, they did not exactly. 30-30s every day for six weeks. Oh, rough. Yeah, not advisable. But anyway, okay, so to complete the picture here, so the authors wanted to also check the expression of an HIF target gene to see if the repressed activity is actually having an effect. Because like we just saw, you know, mRNA to protein relationship is not one-to-one. It's not like you just increase the mRNA and suddenly you've got a concomitant amount of protein increase. The regulation of all these steps is very complicated. I don't even want to tell you actually the number of papers that I've ignored because they just look at PGC-1α mRNA expression between two different training interventions. What? Like, does that mean one is better than the other? I've never thought that. And so I've had to ignore a lot of really interesting training protocols because I think the outcome measures weren't very good, unfortunately. Well, to me anyway, that's my judgment. And so I understand why they would measure that because it's pretty simple compared to a lot of other things you could measure. So not to throw shade at the authors. This is really illustrating a good point, which is that the relationship between the activity of the thing that senses the stress and mobilizes gene expression to create the adaptation to the mRNAs from that gene transcription to the protein, and then even the activity of that protein itself. This is a multi-layered process, and there is a lot of ways for it to... get more active or less active. And I think another interesting thing to consider is that actually when you look at other studies that show like a 5% increase of mRNA or something like that or 5% increase of whatever and it's reported as being, well, it's statistically significant, sure, but is it phenotypically impactful is a big question because we were just looking at protein changes here. Like we're looking at 160% minimum, if not like fully like 250 to 500% increases between like untrained and trained. So what is really phenotypically impactful and measurable, that's a very tough question to answer. Right. I think another interesting thing about that is like, you know, people, if you've ever had blood work done, right, you can measure all these sorts of different things in blood work and they give you a normal range and that normal range may span, you know, a factor of 2, 3, 4, 5, maybe 10, depending on exactly what it is, of whatever arbitrary units. And as long as that number is within that range, your doctor's like, yeah, it's fine. So saying you can, yeah, sure, that test, they can measure a single percentage points probably changing, and you'd be like, okay, but it's not clear that those single percentage points changing actually mean anything. Right, exactly. and that could be for big things like hormone levels where you think like, oh man, my hormone, you know, but I don't know, I feel like there's lots of, there's some, there's a, like an aging clinic master's research joke in there, but like, you know, they think, oh, these, you know, these numbers are really important. You're like, are they? Like, as long as you're- And also, are you having symptoms? Yeah, yeah, exactly. Like, do you feel bad? Because that's, that's your phenotypically significant aspect, right? If you have a- Other symptoms that you notice, and therefore it's like a diagnosable disease or something like that, not just like, oh, you know. Yeah. Number go up. Number go up to any magnitude equals good. Yeah. Right. So... This is why these authors, I think this is a really good study for this reason especially. And when I saw this, I was like, oh, thank God they did this. Otherwise, I would have to go, oh, we don't know. So they checked PDK-1 levels in their cross-sectional study. So what they did was they checked the PDK-1 mRNA levels in the moderately active and in the endurance trained. people. And the thing that they found was that there was a very, very large difference. So they found that the moderately active people had three to four times more PDK1 mRNA than endurance trained people, but they did not check their protein levels, unfortunately. But still, the author's note, as expected, the big difference in PDK1 mRNA levels are... basically also supporting the notion that there's reduced HIF-1 activity in elite athletes. So there you go. There's the entire study. Pretty short one today, but I hope it was interesting and illustrative of kind of what we've been trying to get at, which is like how do the endurance adaptations actually change with time as you get more trained? Like how does that feedback cycle, how does that feedback loop Modify itself as you get better and better and better trained. Yeah, I do wonder here too, like, is this maybe one reason why there are so many big, big, I don't know, big's the wrong word. There's the trend for like, oh, you know, you take moderately, trained or untrained people and you have them do 30-30s or Tabatas or whatever and you see lots of gains. You know, and we said before, like a lot of that could be noob gains and things like that. But man, you want to be hypoxic, that's one way to do it. Like really intense. Like we said, like this here, they're even doing training that you wouldn't really think that you're going too hypoxic in. and still seeing changes. Yeah. But imagine you're driving that anaerobic work really, really hard. And then maybe the, because the, maybe the downregulation might happen faster, totally spitballing, you know, but you imagine that it's clearly happening. Yeah. That could be a follow-up too, is does it happen faster, more faster? if you do mostly anaerobic training. Yeah, I mean, there's a ton of studies and meta-reviews that kind of bear that out, which is that kind of the harder you train, the greater the magnitude of adaptations, even if it asymptotically gets to the same point, and moderate training just kind of gets you there later. And as long as you keep progressively overloading it to some point, because obviously you... cannot just... Yeah, just do the same thing over and over and over, the same workout every day. Yeah, right. And so, and that's another thing that some studies have done previously very well and some others have not done so well and probably for an ease of, you know, ease of the participants. Like if, like in this study, they didn't progressively overload over six weeks. They were just like, yeah, just keep doing it the same target, whatever, it's fine. You know, you're not... This isn't like a study that's looking at what should Olympic athletes do. This is a study looking at how does this change over time, and we don't need the world's best training protocol to actually illustrate that. HAF1-alpha is an active area of research. I mean, it's barely active, but there is some activity. And I think that there's just not a lot that's known about the magnitude of adaptations over long durations or in very highly trained people. If we had Tadei Pigachard do five-minute efforts, what does his HIF-1 signaling look like in skeletal muscle? We're just not going to know. Or the average person who's very well-trained also, I would expect it to be roughly the same. So I think that most of the research done so far just shows that it's in part responsible for new gains in muscles, especially glycogen, glucose, metabolism, iron, nitric oxide pathways. and obviously in terms of capillary density as well. And we do have one paper looking at one-minute efforts. The title is Intense Intermittent Exercise Provides Weak Stimulus for Vascular Endothelial Growth Factor Secretion and Capillary Growth in Skeletal Muscle, right? So like one minute too short. You probably want to look at two to three minutes and longer and especially for less trained people. lower intensity is totally fine. You get all-around benefit, especially if you are newer to training. You do not need to big brain any of this kind of stuff. And actually, a lot of this stuff is pointing to you don't even need to big brain it if you're well-trained either. I also think it's possible, if not probable, that there are some improvements being made in really well-trained people, but I think the effect size would be so small that we're going to need just a ton of data. or a super, super long study, like longer than just a couple weeks. So I'd really like to just see people do like one five-minute effort or a couple and just take a biopsy afterwards and tell me what's the difference between highly trained people and untrained people. That's hopefully an easy one to do. There you go. Yeah, that would be super interesting. And it would only take you a day. It wouldn't take you six weeks. Right, yeah. Cross-sectional study. Do it. Let's see. In all of this so far, I think, well, I don't think I know, but we've actually been ignoring the parallel adaptations that are happening via like PGC-1α, for instance, which expands the mitochondrial reticulum at various intensities. And there's tons and tons and tons and tons of papers on that. In fact, to the point where we have tons of meta-analyses on that. And one of them... which we actually looked at AMPK, we looked at in depth in the AMPK episode. They looked at mitochondrial mass-specific respiration. I don't remember if we got into it in that episode. I don't think we did. But figure four looks at mitochondrial mass-specific respiration. So it's basically VO2 max of isolated mitochondria. So you're looking at something tiny like femtoliters per minute per like microgram or something. Maybe that's not the exact unit, but it's on that order of magnitude. And they also looked at electronic transport chain complexes 1 and 2 expression, and they're most impacted and improved, the expression of them, by sprint interval training, more so than high-intensity training and just kind of middle-intensity continuous training, MICT, as they call it, which basically threshold, sweet spot, tempo-type efforts. And that's in contrast to the papers like the one we mentioned earlier that showed a downregulation of complex four in response to high-intensity exercise. So like, is it going one way? Is it going the other? I think when we step back, all of the performance data that we have says that it's a net positive for sure. But anyway, that's me speculating. So we'll come back to speculation in a second because I wanted to mention another meta-analysis. which we may or may not do an episode on. I was looking at VO2max, mitochondrial density, capillary density, and the capillary density analysis that we want was actually in the supplemental files. It's always funny when you have to go download the five supplemental files to go find an extra figure with like eight different captions. So basically what they saw was that, well, they saw diminishing returns in a lot of ways with all the aspects that they All the aspects that they measured that had a time course, except they saw that with well-trained people, capillary density and capillary to fiber ratio did not actually improve for the well-trained subjects for any training. Interesting. Yeah. I think logically, if you could logic that out, It would be reasonable to expect, right? Like, you cannot imagine a muscle fiber that is 100% surrounded by cavallaries. Right, then it's not connected. I mean, you can't do that. Right, structurally, you can't. Yeah. Because the outside of the muscle fiber has to connect to other muscle fibers in order to transmit force. Right, right, right. You could set that up on like a... You couldn't even set that up on like a Petri dish or something, right? Like you still have to measure force somehow. Yeah. Yeah. So anyway, so based on all of this, I spent way too long thinking about this, which is one of the reasons that this episode took forever to come out, because I was like, where do I go with this? I actually, so my conclusion so far, based on the evidence, is that I don't yet see any strong suggestions of any monitoring effects of HAF. on the endurance adaptations that we actually want. Which goes to the point of today's paper, negative regulation of HIF and skeletal muscle of elite endurance athletes, a tentative mechanism promoting oxidative metabolism. Promoting oxidative metabolism. So we've been talking about how HIF is going to have the pasture effect and reduce potentially the adaptations that we get from endurance training, basically. But they're saying, like, look, as you get better trained, and that's kind of what their study was looking at, as you get better trained, like, HIF's effects actually get reduced quite a bit. And I think that, in part, it's because, here's me speculating, in part, I think it's because the adaptations that you get early on are alleviated very quickly. Like these are enzymes, right? It doesn't take much to make some enzymes for glycolysis. It takes a lot to make new capillaries. And so I think that when you are looking at long timelines, like timelines that we don't really have a proper study for because it's way too expensive, way too much time, you can have a huge dropout, right? But when we do the cross-sectional analysis like these folks did, you do see a very significant difference. And so I think that that's basically what they were getting at. It's like, look, the adaptations that we want for endurance training, like if we have HAF signaling that's kind of going full blast all the time, we're not going to get the right adaptations that we want. And so this is how evolution has seen fit to change things early on. And then as we get better and better trained, there's a point where we can no longer change things because it's practically not possible for whatever reason. Like in the instance of capillaries, you can't have 100% capillaries surrounding a muscle fiber or you just have a muscle fiber sitting in just like the squishy bed of capillaries, which sounds like a burrito in a spaghetti sandwich, but it's an spaghetti burrito? Something like that. A muscle fiber is kind of a spaghetti burrito anyway. Yeah. Weird, like, ratchety spaghetti burrito. Anyway. Yeah. We don't ever really see a realistic decrease of, you know, mitochondrial volume markers like citrate synthase or volume density for the, you know, TEM studies. We don't see a reduction of VO2 max. I mean, VO2 max is central adaptation anyway. We don't realistically see any measurable or practical downsize of the Pesture effect from HIF. And in highly trained people, there may not be a Pesture effect at all, which I think is incredibly interesting. Yeah, that would be super interesting. And I think that that's actually part of what this pathway illustrates, is that as you get better and better trained... If you want to at least keep the stress going, you have to increase the level of stress that you undergo. And there's a certain point at which, if you're looking at that thing about volume, there's a certain amount of volume that you can realistically do in a week before it gets ridiculous and you can't eat enough. Even if this doesn't provide you with actionable advice about how to plan your next training block, this provides you some... interesting information to think about at least in terms of what kind of what is going on under the hood a little bit and how long does it actually take to see changes, see improvements, see gains, maybe a little bit understand maybe understand a little bit more why noob gains happen and why you can't just keep doing the same workout forever even though that'd be awesome. Miracle training, miracle intervals. So also keep in mind that this is something that is, you know, yeah, like you said, still relatively active field of research, and so they're hedging on some of these, you know, conclusions, and they can't, they don't really want to say, oh, this is definitely the way it is. It's hard to say this is definitely the way it is in anything in science. There are always, there, I won't say always, there are often times where people discover exceptions to the rule or exceptions that prove the rule, things like that. But people are slowly peeling back the covers, I guess, on more and more and more of this stuff, and it just takes time. The unfortunate part is that sometimes something like this comes up where it doesn't actually provide you direct, actionable input, even though we would all love that. So I think that this does provide some actionable input in that it illustrates a couple things in training that I think are useful for people. They've certainly been useful for me in coaching when I've been considering what to do with somebody in terms of how much stress do they need, what's a reasonable amount of expectation of improvement. Having some idea of what's going on under the hood, what are the timelines associated with it, how stringent do we have to be with certain training focuses, at what times, I think that that is all really interesting because what's informing my decisions is kind of a thesis that I had in the last episode, in the last Wattstock anyway, which was that how well trained you are should always inform the type of training that you do. and what expectations for performance that you would expect to get out of them, right? So like a good example is FTP work. So if you just do FTP work forever, is your FTP going to go up forever? No, it's not. I mean, FTP work will improve you to max early on, but it won't always. And so when that stops happening, you... absolutely have to do some kind of higher intensity training in order to raise your VO2 max because that's going to limit the amount of oxygen you can deliver to your muscles. And so at some point, FTP work is going to increase your TTE, but it's not going to increase your FTP. So understanding that relationship matters a lot. And that's a very practical performance-oriented one. And so when we're considering adaptive pathways, I mean, you know, the... When we consider how intertwined they are in the regulatory mechanisms and stuff, it's like, maybe we shouldn't consider this. But I think it does illustrate the point that it's kind of the same for all pathways. Like we're talking about the feedback mechanism. The adaptations that you make from the stressful conditions will alleviate the stressful conditions. In some ways, you need to increase the stress, but in other ways, there's not really any practical way to increase the stress. So there's one other thing to consider, which is the potential for utilizing near-infrared spectroscopy, so NIRS. And I'm sure that the reason I only bring this up is because... I think there's probably a lot of people here considering what if I use NEARS to monitor my oxygen desaturation, which should approximate the oxygen tension in my muscle. And so if I get out there and I do a bunch of really desaturating work, is that going to get me some extra gains? And so I think we've already discussed the differential response between... untrained, moderately trained, and well-trained people. So if you're very well-trained, it does not seem like this is going to get you any extra bonus points. And also, if you're not very well-trained, the question is really, what is actually going to give you some adaptation? It's the same question as if you start a training program and you just start doing Any kind of endurance pace riding or tempo or threshold, whatever, you're going to get some pretty robust muscular and systemic improvements kind of across the board. And you can do the exact same thing as a very well-trained person and get absolutely nothing out of it. And so that's one of the big questions here is like where really is somebody's personal... Point of, okay, we're stimulating a pathway a lot, kind of to a moderate degree, or not very much. Obviously, it's a continuous variable. It's not three categorical things. But the point is that I am... So that would actually, in terms of practical utility here, make it really difficult because you would need to know where you respond best personally. So there's that first. I think second is that... You know, the potential effects here of working large motor units may or may not have any utility here. Because I think, you know, if you do a bunch of high-intensity work, obviously you're recruiting large motor units, and is that a way to shortcut some kind of gains? You know, list your favorite hypothetical improvement here that everybody's heard about at this point. I honestly don't think so. Especially when it comes to hypoxia reducible factor. And the reason for this, I'll tell you a piece of data and I'll tell you a piece of speculation. So the piece of data is that oxygen saturation does not really change with cadence. It'll change with intensity, but it doesn't really change with cadence. And by the way, a shout out to Jim Arnold, who's got a great blog post on that, that I actually, I was looking for. I was questioning, you know, what is the actual effective cadence here? And it doesn't seem like there's much. And it's, you know, Jem has a good reference up there for that. It's pretty much straight across the board. So, you know, if we plot cadence on the X-axis and desaturation on the Y-axis, straight across the board, pretty much. So that's the piece of data. The speculation is that when we consider oxygen diffusion in the muscle, We have a kind of a mosaic, if you will. Like if you take a muscle biopsy and you slice it in half the long way so you can see the rings on the tree as it were. Sorry, that's not the long way. That's the short way, regardless. So if you slice it like that, everybody kind of knows what that looks like. You see a bunch of muscle fibers stuck together and then you see a bunch of little capillaries kind of in between. And so when you consider what happens when you are exercising at very high intensity, the recruited muscle fibers are going to be sucking down a lot of the oxygen. And just based on pure diffusion, you should also see the oxygen. It's not like, you know, like imagine you've got a Type IIX muscle fiber in between a bunch of Type I and IIAs that are being recruited. That Type IIx muscle fiber doesn't have its own personal supply of oxygen. That oxygen is free to diffuse to the other muscle fibers. And so is it going to have higher oxygen tension than any working muscle fibers? Probably. How much? Probably not by a lot, I would guess. And so I think that that's probably one of the other things to consider is that there may not be a motor unit-specific adaptation here. And so when you think about cadence manipulation, when you think about anything else in this regard, I'm not sure that there's a lot of easy quote-unquote hacks that we can actually get. So I think that, yeah, I think that that's... is unfortunately another way in which we cannot big brain this. And again, also we just don't have the data with really well-trained people. And maybe there's an effect somewhere. Maybe there's a manipulation somewhere where we can get some extra benefits. But what is it? Where is it at this point? We honestly just don't know. I think overall, we continue to push forward and see what what is out there. And I do think it was interesting you bring up the fact that how well trained you are and your history of training should inform the type of training that you need to do. And that would seem to go break a bunch of trendy things where like, oh, first off, that sort of breaks the idea that, oh, this new type of training is the best training for everyone. Secondly, it kind of breaks the maybe myth that Oh, whatever the pros are doing, you know, that's the thing you should be doing. Why are the pros doing this now? Like, oh, it's the trendy thing that must be better somehow. Um, and also it, it goes against the idea that there's like, you know, the one true way of, oh, you have to, you know, I don't know, ride around in the, in the little ring the whole winter, that kind of shit. Um, which I think we've talked about plenty before, so, you know, I'm not gonna harp on that too much, but, um, Yeah, there are things going on that you can even measure relatively easily that show that people can be doing the exact same workouts and cannot necessarily expect to get the exact same thing out of it, even though you think that might be the case. Yeah. So, alright, thank you everybody for listening as always and if you would like to reach out for coaching, empiricalcycling at gmail.com or head over to the website and if you want to support the podcast, empiricalcycling.com slash donate because we are ad-free. Thank you for all the donations and if you want to give us a nice rating wherever you listen to iTunes, iTunes, wherever you listen to podcasts, I'm going to go have a coffee after this. So, yeah, Weekend AMAs up in the Instagram at empiricalcycling, go ask a question and I guess we will see you all. all next time. See ya.