Welcome to the Empirical Cycling Podcast. I'm your host, Kolie Moore, and we are once again joined by Kyle Helson. And I want to thank everybody for listening. Please subscribe if you're new here and you like what you're hearing. And if you're a returning listener, you can always support the podcast by sharing the podcast and letting people know that you like it and recommending it. And I've been hearing a lot about that lately, and I really appreciate all of that. You can also give us a nice podcast rating and review wherever you listen to podcasts. And you can donate to the show. Things are kind of getting normal back here in Nashville. But if you really want to donate to Hurricane Helene Relief, we would really appreciate that instead of donating to the podcast because we are ad-free. We love off your donations, but we've got a bunch of regular recurring donations that happen. So if we have to live on those for another month or two, that's totally fine. Please donate to Hurricane Helene Relief. And anyway, if you really want to support the podcast, you can always become an Empirical Cycling client. with coaching or consultations. We had a couple spots open up recently, and it would be really nice to fill those spots in because we have some very experienced and excellent coaches who are looking for good people to work with. And so if you feel like you're a good person to work with, then please feel free to reach out to empiricalcycling at gmail.com. And again, as usual, we have... Negotiable Rates for Professional Athletes and for Students because we know that all of you don't make a lot of money and that's cool too. Everybody deserves good coaching, I think. So if you want to ask questions of myself, give me a follow on Instagram at Empirical Cycling and that's my weekend AMAs up in the stories. and if you want to ask questions for the podcast we're not going to do any today because we I kind of want to finish out the series before we get to any of these questions so we're going to skip that for now but normally if we are asking Instagram questions for the podcast so I will ask them over there also so anyway we should start with a refresher of Wattstock 50 not only for Kyle because you were in the middle of the desert when we recorded that one yeah yeah But also because I figure I want these episodes to stand on their own a little bit at least. So let's go over the big takeaways from the last episode. So we talked about hypoxia inducible factor. So this is... Kind of one of the other big canonical pathways in adaptation in the muscles and also in the rest of the body. I think we talked about how it's basically active in almost every single cell type in the body, probably except for like red blood cells and all that kind of non-nucleated things. But it's pretty much everywhere and it pretty much has an effect everywhere. And I think by the end of this episode, you might begin to understand why that might be. What happens is hypoxia-inducible factor, HIF, is activated in response to low oxygen conditions. So we also made a very big point last episode about hypoxic, hypoxic, which is not exactly the same as anaerobic. So just a refresher on that, if you remember our low cereal analogy, Kyle, I think you might appreciate this. This probably looks like your house too, where... You go out and you get a box of cereal but you eat a lot of cereal so like at any given time it looks like there's not much cereal in the house even though there's actually a very high throughput of cereal. That was my analogy because that's the same thing that happens to oxygen. Just because you have a low instantaneous measure of oxygen does not necessarily mean that there's not a lot of oxygen around when the flux is actually very, very high. And that's what happens during very intense exercise, right? Yeah, it's like you're looking at a difference between looking at a value of something or the derivative of something type thing. Derivative meaning rate of change. Yeah. The measurement of the rate of change, the slope of that. So I like to assume not everybody's been through calculus like we have. Because what are we, Kyle? What's the word? Nerds. It's been a while. I miss hearing you call me a nerd. It's good to have you back. All right. So the big adaptations that we get, and now I'm going to start talking near and long term. Big adaptations we get from hypoxia reducible factor being activated in the near term. Well, kind of in the long term, this is what they're supposed to help alleviate anyway too. They're supposed to alleviate the conditions caused by low oxygen, right? So when we have less oxygen, we cannot make as much ATP aerobically through the electron transport chain as we might normally be able to do. So how do we solve this problem in the short term and eventually in the long term? So in the short term, we get an increase in glycolytic enzymes. So we're going to increase the amount of proteins that are involved in glycolysis pathways. So we also are going to increase the amount of glucose transport proteins that we send to the cell surface to bring glucose into the cell because obviously if we're going to have more glycolysis, we need more substrate to use. And what better place to get it than from the bloodstream? All this stuff helps with the cellular energy state, the cell's ability to actually perform work with ATP. And in the longer term, we get a big increase in VEGF. So that's the vascular endothelial growth factor, which we're going to look at in depth in the future. It's kind of the big signaling pathway to make new capillaries and blood vessels and stuff like that. So it's a slower process, a much slower process than just making glycolytic enzymes, which is going to happen in a couple hours to a couple days. So in the last episode, we actually saw over the course of a couple weeks of training in Mice, we saw a couple things drop off pretty quickly. And some of it was like glycolysis, gluconeogenesis genes, and the glucose transport genes, they all kind of fell off between the beginning and the end of that relatively medium short length study. But we saw that the VEGF expression actually stayed quite elevated. from the beginning to the end comparatively by the training that they were doing. And so that's more of a long-term thing because in the long-term, we want to increase the delivery of oxygen to actually alleviate the condition instead of just throwing a Band-Aid on it, which is kind of what glycolysis does, right? Yeah, it's interesting because that kind of sounds like this is something outside of exercise where if you're a person who lives at high altitude, right, that this is the sort of thing that, yeah, historically if you're evolving and you want to not get run down by, You know, the other animals that you're fighting for your food, it might be helpful to have more capillaries and blood vessels to deliver blood to your arms and legs. Yeah, exactly. And that's one of the reasons that we have HIF in almost all of our tissues is because all tissues have to be able to adapt to these conditions because obviously if we are... doing very intense exercise. Obviously, we're going to have low oxygen delivery to most or all of our tissues in the body. But we are also going to have to do something about that. And so the stuff that we're going to be looking at today and a little bit in the next episode are going to be kind of the short-term adaptations where I, well, let's put it this way. I'm very excited. I'm usually excited for Wastok episodes. I know. I'm sorry. But this one's also really, really cool because there's, there's kind of a very, very unexpected result about what's going on. So I think last episode I talked about how there's some weird, there was some weird stuff happening. Like there was a decrease in expression of the, I think the cytochrome C oxidase. Protein, but something like that, or regulatory protein involved in that. So we're going to be looking a lot into that specifically today. But anyway, so for a little more background, for well-trained people, HIF is actually activated at, sorry, for less well-trained people. So for people just off the couch, if you are just undertaking a training program, or if you are kind of a young mouse, You're, you know, I don't know, 20-something weeks old. Let's say you're a hale young mouse and you're ready to run on the treadmill for science. Yeah. Yeah. Science. I'm sure that's exactly what those mice are thinking. You are going to actually get a lot of HIF activation and it's going to happen at a lot of intensities. And so that's one of the things that we're going to see in this paper today in mice who are basically... kind of running a threshold more or less. And so the activation of HIF actually reduces at more moderate intensities as you get better trained and as you develop more capillaries to alleviate the low oxygen conditions, you can deliver a little more oxygen that way. So as the adaptations happen, the... near-term adaptations are going to reduce in magnitude, right? Because the primary function of the exercise-induced adaptation is to decrease the homeostatic perturbance that we're experiencing. Kyle, can you please say that in English? Yeah. Yeah, so your body wants to keep things like your blood pressure and your core temperature and, I don't know, your blood oxygen levels at pretty much the same level all the time, like the level that it needs to keep you Comfortably alive, but it doesn't want to work more than it has to at any given time. And doesn't also want to keep you like teetering on the brink of death. So your body is, you know, tries to keep, and this is maybe why people see early noob gains. They get really fast adaptations early on to, you know, brand new exercise because your body's like, oh man, we haven't seen this before. We're going to try to make sure this person doesn't die the next time they try to do this. Obviously, your body doesn't really know that you're doing this for, let's call it fun, or you're doing it with the greater goal of winning a bike race. Right, yeah. Oh, also, I totally forgot. I thought the last episode was in mice. It just came to me. Sorry, we're recording this a little late in the day. So my brain is a little bit slow on the recall, but I scrolled up to where we had the notes for the other one. And guess what? It was in untrained cyclists. Ah, even better. So they were thinking for science. For science, yeah. Yeah, science. I was thinking of mice because this paper today is about mice. But also the other cool thing that we saw was the... individual responses were actually quite widely varied between people in the study from the last episode. And I thought that that was really cool because that's another thing that you have to watch out for as you get better trained is you need to make sure, I mean, we could probably talk about this in the wrap-up instead, but why not do it now? Because as you get better trained, you've got to make sure that you are doing what you are responding to. Phenotype versus performance is another thing that we're going to talk about today. But I don't want to get too ahead of ourselves because if you are new to training, if you're thinking about the HIF pathway, which most people probably shouldn't, most people probably shouldn't be thinking about anything in terms of this. Other than consistently exercising. Great. Consistently out there, yeah. Yeah, yeah, yeah. The Keep It Simple Stupid philosophy really works for... for most training. Anyway, so these HIF-induced related adaptations are actually going to happen in parallel with your other adaptations. So, you know, a billion studies out there are showing increases in mitochondrial content, VO2 max, and all sorts of other stuff as you go from couch to crit. Is there a couch to crit plan yet? I'm sure somebody has one of those. I'm sure someone's named something like that, yeah. Yeah, if not, yeah, you can have that. Have fun. Go make a couch to crit plan. So another near-term adaptation that we see, we kind of already alluded to this, is a reduction in oxidative gene expression with hypoxia-reducible factor being activated highly. So why would we want this? In conditions of low oxygen, it would make sense for first principles to reduce the reliance on oxygen and oxidative metabolism until the long-term adaptations like capillary density have alleviated these conditions. So we can imagine if you are brand new and you do a threshold workout day one, like you're probably going to get a decent amount of like all sorts of like, holy shit, we're really exercising. We need to... All Hands on Deck, like makes adaptations fast. That's going to happen. It's going to be AMPK. You're going to be looking at sirtuins. You're going to be looking at CAMK. You're going to be looking at HIF. You're going to be looking at all of it. And as you get better trained, let's say for threshold, like these things are going to start to drop off as you build capillary density and mitochondrial density and all that kind of stuff. Other thing to remember with all this in the long term is that how well trained you are kind of should have an effect on how you consider your training because the performance changes you would expect to get from the training will change based on how well you've alleviated the perturbation conditions that you are training. What doesn't matter what kind of interval it is, anaerobic capacity, threshold, VO2 max, it's like, blah, blah, blah, list goes on. So that's probably one of the things to consider, just in general, not even with hypoxia reducible factor, but just in general, what kind of overload do you actually need? And what is the mechanism of the overload? I think in a lot of ways, that's one of the most useful things that all this nerd shit has taught me over the years. That's interesting too because it gives a reason to why you can't just do 2x20 forever and you have to do something more than 2x20. Like a deeper reason other than just more is better. Yes, very, very true, yeah. I mean, and that's one of the reasons that we're going to see in this paper, you know, we should all be considering our performance instead of kind of... Big Braining this stuff. Because I mean, I can't, any number of times I've said previously, I think most people could get good at coaching or coaching themselves by just looking at the performance and seeing, if I do this, my performance gets better. If I do this, my performance gets better. And I think, you know, just in general, a coach is a good way to kind of shortcut that, like, you know, consultation or hiring for coaching, because, you know, we've seen all of this stuff. I think at this point I've coached people through hundreds and hundreds of seasons, maybe a thousand at this point, something like that. So, you know, we've got experience. A thousand? No, maybe a thousand is too much. Anyway, I did the math on another episode. I forgot what it was. So, anyway, let's get to our main paper today because this is one where we're going to actually knock out a hypoxia-inducible factor. So, it's not going to be active in any tissue in the body for these poor mice. So I don't want to give away the game, but we've kind of already done it. So the title of this, HIF-1 Alpha in Endurance Training, Suppression of Oxidative Metabolism. What? Okay. The researchers knocked the HIF-1 Alpha gene out of skeletal muscle from a strain of mice. So that's why we're going to just henceforth call this... the Knockout Paper. And for those of you who need a refresher on the mechanism, HAF-1 beta is constitutively made. And when oxygen drops, HAF-1 alpha, which is normally degraded constitutively, is left alone, and then it dimerizes with HAF-1 beta, and then it goes and does transcription factor things, just like PGC-1 alpha, except for the fact that You need it, well, because the conditions are transient, like low oxygen conditions are transient in the skeletal muscle, like during exercise, you need to have these things ready to go at a moment's notice. And so that's why it's made constitutively and degraded until you need it. Otherwise, you might start to ramp up your... Gene Expression, and like, okay, yeah, we'll have some hypoxia-reducible factor active in about four to eight hours, something like that. Okay, cool. Check back with us. Like, okay, cool. But like seven hours ago, we were done exercising. So what use is it now? Anyway, so what we've got here is four groups of mice. We have normal wild-type mice, trained and untrained. So this is a, I think a... Pretty well done paper because we have a control condition for the knockout and for wild type. And so our experimental condition is mice with hypoxia reducible factor 1 alpha gene knocked out of skeletal muscle. They also have that trained and untrained. So the training groups, they did 30 minutes a day for five days a week for six weeks. That's 30 sessions total. The mice started at 18 meters per minute on a 5% incline. It might be degrees. The paper says percent, but a little later it says degrees. And then they had two recovery days and then they did an endurance test. The endurance test was on a 5 degree incline, not percent. Might be a typo, like I said. I don't know. Ask the researchers, not me. So the test was they started at 12 meters per minute. So previously they did 18 meters per minute. on a 5% incline for 30 minutes a day. So this is their endurance test. Started at 12 meters per minute, increased 4 meters a minute every 5 minutes until the mice reached 28 meters per minute, and then they held it to exhaustion. For those of you playing at home, 28 meters per minute is like... One mile an hour. And for mice, that is flying. So basically, you can think about, they're basically doing kind of an FTP test. And you'll see why I say that in just a second. What they did was they not only looked at performance, but they also looked at a bunch of biopsy characteristics of the muscles in the mice. So they looked at like gastrocnemius, that's a calf, and they also looked at the quad muscles. But that's, we're going to get to that in a minute. The big question is... That's a small tape measure to the... Yes, very small tape measures. So with no HIF, how did the knockout mice fare in terms of adaptation? The untrained wild type control mice did their endurance test at 41 minutes average. So perfect kind of estimate for like a human doing FTP, something like that. 41 minutes, perfect. The trained mice did 67 minutes. Excellent. Very good. The untrained knockout control mice did 44 minutes average. So basically the same as the regular wild type control group. Untrained, yeah. Untrained Wild, yeah. The trained mice did 78 minutes average in their endurance test. So the groups improved 63% and 76% respectively, but there was not a significant difference between these two groups, the two training groups. So statistically speaking, this is a similar to same improvement. Like they're not, the distributions were not such that we could say the knockout group was definitely Better. So all we can really say for sure is they did about the same and that's fine. They're mice. Yeah. That's cool. So. Yeah. If you heard like, you know, the, it's because the averages are slightly different to start. You're like, ah, 67, 78. That's, you know, yeah. Measuring biological, as you said a lot before, measuring biological things is hard. Like they are not nice. Neat Things. Maybe those mice were not mentally feeling, you know, who knows? Yeah, well, I mean, that's just the kind of nature of inherent variability in living things. And, you know, there's probably measurement error, there's probably individual variation, there's probably, you know, a bunch of... Noise thrown in from a bunch of stuff. And so basically what we're trying to do is we know we're going to get some kind of distribution. It's not going to be every animal is going to be the exact same yada yada because like otherwise we would expect the same with people, right? But we get very much not the same thing with people. So what happens with mice is, or in this case, we're looking at, you know, we're looking at a certain distribution centered around 67 minutes for the trained wild type and 78 minutes for the trained knockout mice. And we look at the overlaps and we run some stats and we go, okay, these are basically the same thing. There's no way that we could say that these two are any different because of anything but the inherent variations in the data or something like that. I'm sure some statistician is going to write and be like, you got that all wrong. And I may have. But I think I'm pretty close, right? Yeah, I think that... All right. We've said before, too, like, the standards for measuring biological things, like, you know it's going to be hard, and so just the... You accepted that there's more error and that teasing out what is actually statistically significant can be quite tricky if all you've got is mice. Especially if all you've got is mice, for sure. Like, are you ever going to ask a mouse, like, were you feeling off today? Right, exactly. Yeah, yeah, yeah. There's all these things that you could do with humans that you can't do with non-human subjects. Yeah. Okay, cool. So the, so we have about the same improvement for their performance. So about 40 to, you know, 70-ish minutes for their endurance test, which is great. Okay. So fuel usage is an interesting one because looking at fat, and Carb Oxidation. Whole body, because this is via respiratory exchange ratio, RER. For the wild type mice, this was about half fats and half carbs, 0.85 RER. And when they were trained, it dropped to 0.8, which is with a very significant difference between those two groups. The knockout mice, both trained and untrained, already had an RER of 0.8. And so there was no difference between the trained and untrained group in terms of substrate usage. Interesting. That is weird. Right? I knew you would like this. I'm so stoked now. All right, let's go. Okay, cool. As the paper notes, quote, loss of HIF-1 alpha gives rise to a phenotype mimicking exercise training. Huh. Yeah. First time I read that, it blew my mind because I was like, how could this be? And then I did a lot more reading and I was like, oh yeah, okay, that makes sense. And we've kind of laid the groundwork for it, but we're really going to dig into it this episode and I think the next one too because there are some papers on trained individuals with this stuff. It's really cool. So this is kind of an unexpected result. Why would knocking out an adaptive pathway lead to these mice having a trained muscle phenotype in terms of their mitochondria and fat oxidation and like fiber type and stuff, but without the trained performance. Why do they have to train to improve if their phenotype was already the trained phenotype? Because they... Everything... Yeah, okay. Let's see if they're... All right, put that one under your thinking cap. It's going to get weirder. Okay. So the researchers asked was, is it because of fiber type? And the answer was that they did histochemical staining. They showed that the wild type mice, gastrocnemius, had a 36% shift towards more type I fibers after training. That's the wild type now. and a 17% decrease of Type IIb. That's the super fast switch that humans don't have. And that's standard. That's normal. Like there are any number of studies that see that in wild type mice. The knockout mice had zero shift in fiber type. Zero. Because the Type I fiber percentage was already elevated. So even from the beginning, they don't, so like they're born and they don't have this pathway. They don't have this pathway that responds to low oxygen conditions. Right. So when they... So they... So if they're just existing, even when they would be... Like as you get older, right? Just as you get... As you get sarcopenic. Yeah. Yeah, your body would... You and I are both in the throes of sarcopenia right now, I'm sure. Yeah. Your body would just start to adapt anyway, but now you can't adapt to that pathway. Actually, as we get older, the loss of fast-twitch muscle fibers, nobody's really sure why that happens as far as I know, but I think the most commonly bandied about reason I've seen is disuse. Like, you're not using these fibers, you're going to lose them. which is another reason people should lift weights as they get older. So, okay, yeah. So, okay, hold on to all those thoughts too because we're not done with the weirdness by a long shot. So, we know that part of the targets for HIF pathway is vascularity, right? So, was there a change in capillary density? In wild type mice, They definitely had a significant shift in the deep gastrocnemius muscle in the capillary to fiber ratio. We went from 1.26 to 1.46 average, plus or minus .04 for both, so that's pretty significant right there. Knockout mice, again, no shift in capillary density, but take a wild guess, they already had elevated capillary density comparable to the trained mice. So we got another one. All right. Getting Weirder. Now, one more similar thing in this vein. You already know where this is going. Enzyme activity. Trained wild-type mice improved their HAD activity. That's the enzyme involved in fatty acid breakdown in the mitochondria. The trained knockout mice did not improve it. You know why. They were already elevated. Same thing with citrate synthase, our front door to the Krebs cycle. Raised in training by wild-type mice, already elevated in the knockout mice. Let's start to uncover Yeah So you're saying it'd be interesting because you're like people always talk about some sort of genetically engineered like super athlete humans in the way off distant future. It sounds like this would be your route to having children that it can at least beat up on the children in like t-ball and stuff. You want your kid to be like an amazing kid? I would want to wait for the genotype that gets somebody already at the trained performance. Because we don't have that here. Fair. Yeah, yeah. To me, that was the most mind-blowing thing of all was we've got this phenotype shift, but we still had to train to get the better performance. Right, right, right. Yeah. So, now. We get rid of the low O2 sensing pathway, we end up with a more aerobic phenotype. How? Well, the investigators took the untrained mice and had them do a 30-minute treadmill run at 24 meters a minute and looked at AMPK activation. So do you remember our old buddy AMPK, response to low energy state, you know, elevated AMP in the cell, all that kind of stuff? In the untrained wild type mice, so these are our natty mice, they're on, what would you call it? If they're knockout mice, they're not really enhanced though, they're like de-enhanced, de-hanced. Yeah. So these are our natty mice. For this specific test, this is not the regular endurance test we talked about before. For this one, there was a three-fold increase in active AMPK. In the untrained knockout mice, they were lacking the HAF. AMPK is actually constitutively active at the same level as the just exercised wild type mice. Huh. They come out of the box with already active AMPK compared to wild type mice. Interesting. So this is a very strange thing. And we're going to talk about why. Because Let's think about it from terms of performance. Let's answer our previous question about why getting rid of an adaptive pathway leads to these mice having a pre-trained phenotype, but they still didn't have the trained performance. Since the AMPK was active in the knockout mice, even at rest, it actually shows a dent in the energy state. So the ATP to AMP ratio, you know, the potential work energy that ATP has, if you missed all our previous episodes on all this stuff, just look up ATP mass action ratio, just Google that and it'll explain everything. So this would actually go a huge way towards explaining the pre-trained phenotype that we see. But the but here is that it took the knockout mice still, all of that pre-adaptation, We can call it pre-adaptation, just to be the same performance as untrained wild type mice. Right, right, right. So knockout mice, they have a problem, clearly, with like maintaining energy homeostasis in cells. And so without as much, you know, or without as much HIF active, There's a lot more aerobic metabolism that needs to be done just to do even a half-assed job at being untrained. Yeah, that makes sense. They're like, they, obviously, it's not like some of the papers you've read where basically they're entirely, they cannot be anything but entirely sedentary because of the gene knockout, but they're, but, you know, in order to They basically have this, because they have this deficiency, they're starting a few squares back, and so they're already working to overcome this, it's not an adaptation, but yeah, this deficiency. Yeah, yeah, because like if their cells were doing a whole ass job at maintaining energy state, you wouldn't see, you would see the wild type AMPK activation, and I'm sure if they looked at If they looked at other adaptive pathways like CAMK and like sirtuins and things like that, then they may see similar. They may not. I would be very interested in that result. So I think what this all points at is that there's a lot more to performance than just in your muscles. and there's multiple potential explanations for why knockout mice ended up increasing their performance none of which the researchers really tested for because it was beyond the scope of their hypothesis testing but in the future I would have loved to have seen additional testing like do some VO2 max testing do some TEM on their skeletal muscle, mitochondria, things like that. I would have loved to have seen that. But I think the biggest takeaway here in all of this is that just because the phenotype suggests that a subject should have a certain performance does not necessarily follow that the subject's performance will actually reflect that phenotype. or what that phenotype would make you think their performance would be. So we can, so like, oh yeah, sorry, go ahead. Oh no, that's interesting, but that's kind of like the, you know, if you're, if you're thinking like, oh, it's like, what is this relevant? But that does remind me of that, like, oh, you know, you can get, plenty of people can, could, if they did nothing but train, get to say, you know, I don't know. Median Cat II Cyclist Fitness, but that doesn't mean you could hack it in a Cat II crit type thing. You can get there and like, yeah, they express this phenotype, but it is not the same as just one element. Yeah. Well, here's an example. It's probably a dumb example, but it's an example. Of all the examples, here's one. If you take a cross-country skier with a massive VO2 max and you put them on a bike. Are they going to have good performance? Oftentimes, you know, they're going to be way better than the untrained amateur. They're going to be way better than even a trained amateur a lot of ways. But are they going to be similarly world class? Almost always the answer is no. I think we've seen that enough times with semi-high profile examples of people, you know, big teams signing, oh, this guy's got a, he blew a 98 VO2 max. Wow, let's give him a contract and yeah. Anyway, so what we're seeing so far is that the muscle phenotype compensates for when HAF1-alpha is knocked out, and probably with our familiar adaptive pathway, AMPK, that we're already familiar with, sensitive to energy state. But there's still something weird going on here, and I want to dig into it a little bit because it's part of this paper, and this is going to serve as a preview for, I think, probably the next episode. One of the other things that these researchers did as kind of a parallel is that they also took cultured primary myoblasts. Think about like muscle stem cells and solution basically. They knocked out the HAF1-alpha again, kind of like in the mice, and they used wild type cells as control. Then they looked at oxygen consumption in normoxia and hypoxia. Kyle, you're going to love this. I can already tell. Ready? Under hypoxic conditions, wild-type cells went from consuming six nanomole per minute per million cells. Doesn't matter. Just think about six. If you have two max of six, let's say. Yeah, six in arbitrary units. Yeah, six in arbitrary units. But then they went down to... 2.5 nanomoles per minute per million cells. So 6 to 2.5 arbitrary VO2 units. So from normoxic to hypoxic conditions. The knockout cells consumed the same amount of O2 in both conditions. And this greatly stunted their growth. Why would that happen? If you've got cells, let's set this up, just so everybody's clear, you've got knockout cells consuming the same O2 in hypoxic conditions as normoxic conditions. Let's say it's like 5% or 10% as much as normal oxygen pressure. Why would that stunt your growth as a cell? Just because you have less available... Resources all the time. You have the same amount of resources all the time, and that number is small. Yeah, right. So let's use our cereal analogy. How much more fat could I possibly get by eating the 100 grams of cereal I have left in the house? I would need a lot more than that. And so my efforts to grow my belly... is going to be very stymied by what little cereal I have. I clearly need to make a grocery run like tomorrow. It's like if you're buying the regular box of cereal at the store versus buying the Costco pack, right? And you go to the store the same number of times a week. Let's call it once. Right. Yeah, let's pretend it's once. Yeah. Yeah. Okay, so... What's happening here with these cells is that the wild type cells are successfully keeping up normal growth rates because they're adjusting their preferred metabolic pathway according to conditions using hypoxia inducible factor. And so when we get a knockout cell that cannot make this shift, it's basically trying to get fat on low cereal, but there's not enough cereal around so it can't grow. And so the wild type cells went, oh, there's no cereal around. Well, I've got ice cream. Cool, I'll have ice cream. So they grew on ice cream. Ice cream meaning like, you know, glucose through glycolysis. So this is called the Pasteur effect, named by... Somebody, I forgot to Google him. Who's the Pasteur effect discovered by? No, it's by Louis Pasteur, of course. So the Pasteur effect is where cells consume less oxygen in hypoxic slash anaerobic conditions and perform more glycolysis. And for organisms that can switch back and forth, think some bacteria, yeast, things like that, that means that they will make alcohol. as their final electron acceptor instead of going through the electron transport chain. So in living cells like yeast, pasture effect increases their adaptability to different environments. Although I did learn when I was looking this up that baker's yeast, or not baker's yeast, sorry, brewer's yeast can actually do both under normoxic conditions. So you don't have to like suck all the oxygen out of the thing to make them ferment, which I thought was cool. Anyway, so this ability to use hypoxia-inducible factor pathway means cells are much more flexible in their ability to do what living organisms do, replicate, survive until conditions improve, play Xbox, ride their bikes, you know, well, I'm just kidding. Anyway, so HIF, Well, HF1-alpha specifically, since that's our knockout, it's a key modulator of the Pasteur effect, which, as mentioned, not only increases expression of enzymes for glycolysis, but it also down-regulates activity at key points in the electron transport chain, like complex four, like where all those electrons finally become water, and in this knockout study, or similarly, it down-regulates activity of the conversion of... Things like pyruvate to acetyl-CoA. So that's our glycolysis into the Krebs cycle thing. So protective mechanism, right? We don't have a lot of oxygen. We want to make sure that we're using less oxidative metabolism. So because, you know, you don't want to be like those knockout cells that just couldn't grow and pretend like everything's fine in an environment of low oxygen. You don't want to be sitting there like, like, let's say you're in a spaceship, right? I'm sure we've all... played this mental game of like what, you know, you're watching a sci-fi movie and you're on a spaceship and like something happens and they're like, okay, we only had this much oxygen left. Are you going to jump on a treadmill at that moment? Yes, because I hate everyone. And myself, I'm taking you all down with me. Yeah, so that's what this is like. It's like, it's like if you, if you're low on cereal, it's like pretending like that's the only food you've got in your house. I mean, if that's the only food you have in your house, please go to the grocery store, turn this off right now. So HAF1 also regulates expression of some of those key genes plus the genes behind the key regulatory proteins. So we're talking things like... Like pyruvate dehydrogenase kinase, which modulates the activity of the pyruvate dehydrogenase complex. So things like that. And there's a lot of other studies, and we could get into gory detail, but believe me, we're pulling back on that stuff at the moment, that show HAF1-alpha knockout in hypoxic conditions really stunts growth of cells and culture. I mean, we kind of see in this paper, but there's like a lot more detail that we could go into. Anyway, I may or may not link another paper in the show notes. Anyway, so when HIF is active, it engages the pester effect, which will reduce the cell's reliance on aerobic pathways because, duh, low oxygen. And it's going to increase the cell's ability to... Do things like exercise, survive, replicate, etc. Well, not that like yeast and bacteria exercise, but you know what I mean. So while it's great to keep things like bacteria or even tumor cells growing like wildfire, our actual, as humans and larger organisms, our energetic needs are way too big. So we as larger organisms, and people like me as very large organisms, and we use HIF to respond to more transient needs, like VO2 max intervals, or Kyle, you've been to Machu Picchu? That's way up there, right? Like, you know, short or even long trips to altitude, like this pathway gets activated. Yeah. I mean, that's probably why people write, like, even after a few days, a week, two weeks, in a place with a high altitude, low oxygen, you've adapted somewhat. Like, it would be bad if it took your body months and months and months. Yes. Like, oh, sorry. You just moved to Denver? Well, you're going to be half a person for three months. And, you know, what's funny is that kind of stuff is not necessarily even in the muscles until you start exercising at that point. At that point, you know, the HIF activation is actually in your kidneys, mostly in your kidneys. And it makes EPO, EPO, or thropoietin in response. So that way, your bone marrow goes, oh, more red blood cells, cool, we can do that. And so that's another reason that, you know, a lot of times that really well-trained people who are looking for more adaptations in that way. Go to altitude for months and months and months and months and months of the year. So any other thoughts before we kind of wrap this up? No, I think this is interesting because I feel like a lot of times when we look at the knockout mice, they do not do well. No, they don't. They live very sad, short lives. And at least here, it makes sense from an evolutionary standpoint, like you have more than one energy pathway so that it makes sense. this wouldn't totally ruin them in terms of like they just can't do anything. It also is interesting though that it's like yeah you're because you start it's basically you started from you know three squares back so you had to you had to basically behave like you had already been training just to get to square zero where the other mice started and so your body was working or their bodies were working this other pathway hard because that was their only choice. They had no way to be able to sense or adapt to this, you know, normal, just growing and developing the stimulation that their bodies would get from normal growth and development. And that's why they even started, you know, untrained, quote unquote, untrained with a phenotype like the trained. Yeah. Yeah, and I think that this paper really showed that there's, because this was in 2007, I think, so what are we, that's 17 years old now, this paper, almost 18, geez. So damn near 20 years ago, this paper came out linking you know activation of hypoxia inducible factor pathway to down regulation of aerobic metabolism and kind of showing with cells and culture the kind of base principles of why that might be and you know now we as you know more more you know educated I guess we could say or more well researched or more you know there's been a lot more research on this in the subsequent decades. So let's put it that way. And so we have the ability to go, okay, well, obviously this is only for small things. And, you know, what happens in cell culture, I guess, stays in cell culture because it clearly doesn't happen in mice where, you know, you can't, you can't, like, you cannot put mice in a no oxygen, you can't put mice. in an experimental condition with no oxygen and be like, HIF-1 alpha is going to activate and they're going to do just fine in glycolysis for like months. Speaking of which, Kyle, I don't know that science has ever proved that we need oxygen to live. Can we take some people and have an experimental no oxygen condition? I'm going to need to see this. There's probably one or two like... Research Ethics Committees that might have some questions for you if you propose this. You're probably right. I bet somebody did it in like the 1600s, like right after they discovered oxygen or something. Anyway, so yeah, us as much larger beings, like... We are not cells in culture. But yeah, like as we start to do this, as we get off the couch or we start to do higher intensity training as trained people, we are going to have, we're creating conditions that become alleviated by the adaptations that we make. And this is kind of the fundamental point of all. Phenotype Changes. We are trying to change our phenotype in response to a stimulus that we are giving ourselves. Some might call it fun. Cells might see it as a noxious stimulus. Like, oh my god, how come you're always fucking up my energy state, man? So as we train, we get better at withstanding these conditions. That's why both absolute and relative performance Improves. How long you can hold your FTP. There's a relative performance aspect that improves. How much your VAT max goes up. There's an absolute performance aspect that improves. All these kinds of things happen. And as we get better trained, the kind of training that we respond to changes as we go. And I mean, this is one of the reasons that like, you know, there's like so many worlds where people stay at altitude. they want to improve all of these other metrics and I know that there are some very well-known examples of people who refuse to go to altitude and are going just fine since but I'm I don't know, has somebody won a grand tour in the last like 10 years who has not spent a lot of time at altitude? I'm not entirely sure. I have no idea. That'd be interesting because I feel like more recently it's been easier to kind of see what People do in the offseason, you know, with social media and Strava and all this stuff. And I feel like even 10 years ago, it was much harder to know exactly when and where teams are doing training camps and things like that, right? Now you have friggin', you know, Wout uploads his stuff to like Strava. So you can just see what, you know. Maybe not all of it, but you can see generally, oh, they're here now, and then they're going here next. So it's interesting. Yeah. What I would really like to know is what's the last example of a world tour athlete winning a grand tour who didn't go to altitude but also was not doping? I mean, I think that's when the altitude thing became popular, when you had to make your own endogenous EPO by going to altitude instead of just injecting it. I wonder how related that is. Probably very. Anyway, so the other point of what we've been talking about today is that just because, again, because a phenotype suggests a certain level of performance does not necessarily mean that the performance will actually reflect having been trained. So fiber type, mitochondrial content, Citrate Synthase Activity, HAD Activity, what are the other ones? You can be 6'7", but it doesn't mean that you're good at basketball. Yeah, isn't Wingspan more predictive of basketball success than actual strict height? Maybe, but I know after a certain point, like, you know, like... 90% or some very high percentage of people who are 7 feet tall have played in the NBA, right? There is a certain height that once you are, they're finding you. First off, there aren't that many 7 feet tall people. One of my old friends is like 6 foot 7, 6 foot 8 or something like that. And he's an outlier, let's put it that way. But he's an excellent programmer. Anyway, so I think those are the big takeaways. So next episode, we're going to dig into HAF activation and regulation and the interaction between the regulation of aerobic pathways in well-trained athletes. And then I think after that, we will get into capillary formation because that's very intimidating because there's a ton on capillary formation in the literature. And a lot of it actually has to do with cancer. Whee! Nice, cheery subject. But that's actually one of the reasons. I was thinking about doing like, what happens if we knock out a regulatory protein for HAF paper? and all of them are very sad. If you knock out some regulatory proteins, it's lethal to the organism. Those mice just ain't going to make it. If you knock out others, like they just grow a lot of tumors. So in those papers, not a lot of exercise gets done. I was thinking about doing something like that in terms of showing the cellular phenotype change, but I think we've kind of... We figured it all out on this one, so we have a happy group of mice that are potentially, hopefully, living just as long as the wild-type mice in that group. But anyway, after that I want to look at the kidneys and central regulation of VO2 max and because we kind of talked about improving it previously but I also want to look at the regulation and that's also going to talk about like untrained versus trained people and there's going to be a lot more in terms of that coming because I saw a couple papers I'm really excited to bring to everybody and some of it's just not good news. Do you want to know the spread of untrained VO2 max in men? We're going to find out in a future episode of Wattstock. What is it? Is it like? Don't even ask. 20 to 70 or something insulting like that? You heard it here first. No, it pretty much is like 20 to 70, something like that. So imagine being somebody who starts with a 70 VO2 max and like, oh, I wonder if I can improve it up to 80. All right, cool. Have fun with your pro contract. Anyway. Yeah, seriously. Yeah. That's that person who you're like, oh, yeah, we just ran the mile for fun in elementary school and this person ran like a sub five minute mile. Sixth grader doing sub fives. Oh my God. Yeah. Yeah. Anyway, all right. Thanks everybody for listening. Really, really happy to have Kyle back for our Wattstock and we're going to have, we'll probably have the next one hopefully in the next month or so as I kind of settle down back from getting back from the hurricane. damn near caught up on an entire month of calls. So thanks everybody for your patience with all that stuff. And yeah, anyway, so our usual outro stuff. So if you like the podcast, please subscribe. And if you want to share it, please share it with some friends, recommend it, put on some forums, all that kind of good stuff. Thanks for all of that. And if you really want to support the show, we're ad-free. Well, donate to Hurricane Helene Relief instead for a little while anyway. But if you want to become a coaching or consultation client, please shoot me an email at empiricalcycling at gmail.com and let me know what you're thinking about and we are happy to work with you if we possibly can. And otherwise, just give me a follow on Instagram. We can AMA's up in the stories and I will try to remember to post some memes. Anyway, happy Halloween, everybody. Bye. Have a good one.