Welcome to the Empirical Cycling Podcast. I'm your host, Kolie Moore, joined as always by my co-host, Kyle Helson. Thank you everybody for listening as always. And if you are new here, please consider subscribing to the podcast. And if you are coming back, then thanks for coming back. We really appreciate you. And if you want to support the podcast, please go to empiricalcycling.com slash donate because we are ad-free and that is a way that you can support the show and keep the lights on here. We can also, we also benefit from you sharing the podcast and we appreciate when everybody shares the podcast and says, Check this out. Friends, forums, family, if you have some cyclists in your family, really appreciate all of that. Been hearing some really nice stuff lately. So thanks again, everybody, for all of that. And if you want to become a coaching client, we're going to get into kind of, this is all going to tie into our coaching philosophy near the end of this. So we're going to talk about that. And if it sounds like this is a place that you want to work with us or you want to consult with me or one of our coaches, shoot me an email at empiricalcycling at gmail.com because we coach cyclists of all levels and we have varying rates and we can do consultations, look at your data and plan out your season if you want to keep coaching yourself. So if you want to Ask a question for the podcast. Follow me on Instagram at Empirical Cycling. And if you want to participate in the weekend AMAs, we just finished a round of those. Please go follow me there too. I post one every weekend. And that's also where I post for Instagram questions for the podcast. I already said that. Okay, moving on. So this episode is gonna try to integrate and wrap up a lot of the aerobic... Adaptation Signals that we've been talking about in the muscles. And so we're going to go very, very deep on our main culprit, PGC1 Alpha, and we're going to try to... talk about some aspects of PGC-1 Alpha that usually don't get discussed in training literature and in a lot of media. So I'm far from the first person to read this research or talk about it. But if you are here, then I figure you probably want to hear it because this episode is going to be... You're a nerd. Because I'm a nerd. And this is going to be a typical Wattstock episode, but only a little bit more so. Partly because of the length. So we are like a handful of minutes in and we have many, many, many pages of notes, way more than usual. And we also got some really excellent questions from our listeners on PGC-1 Alpha. So we're going to try to think about aerobic adaptation, like broadly speaking, and also kind of the nitty gritty. Because generally speaking, When we talk about aerobic adaptation, we're talking about broadly one of two things. We're talking about cardiovascular or we're talking about muscular. And so we've been focused on the muscles for the last few Wattstock episodes and we're going to move to the cardiovascular stuff in the next few. And we've got some really, really, really cool stuff coming up. And so I'm really excited to get to that. And we're going to get back to VO2 max and plasma volume because I don't think we've really talked about it too much on the podcast since 20... 20-ish? So it's been a little while. But let's take a step back first and think about the past few episodes and kind of what we are integrating, I guess we could say. Like PGC-1 Alpha integrates a lot of signals. So Kyle, I think you missed like the AMPK episode, maybe the Redox episode, but you kind of know generally what's up with this. So like what have we covered so far in terms of aerobic adaptation and training and stress and all that kind of stuff? Yeah, so I think we've just kind of been looking at where all of this, what is actually the core mechanism behind, you know, you going out riding your bike and then you getting fitter and we've taken a lot of cracks at like what it isn't as well, you know. Typical fashion. But I think, yeah, I think the kind of the goal of the series is just to say, okay, So why actually are you getting fitter or what are the signaling pathways and what are the actual important parts and consequences of training and how can you take that information to better inform more training later, right? Like if you have a misconception, for example, on how aerobic adaptations occur, you might think that say some latest trend in fitness or training is you know the secret the silver bullet to you getting fitter and then you realize later that oh like for example with the high intensity interval training studies like oh but then it only works on untrained people because of the mechanism that's actually behind it even though it was briefly touted as you know the latest and greatest whatever. Yeah and that's actually something that we're going to get to in this podcast too is kind of discussing what works on who and kind of taking a step back and because now we're at you know we're at kind of the end of this section of uh of this series where we're going to be thinking about what can we really take away from this and that's part of why this episode is going to be so long is because we're going to be really breaking it down in terms of like what can we use this for how do we interpret studies based on all this stuff and all that kind of stuff yeah many hours later yeah and so I think I think that's an important tool too just because kind of in the same way that If you're teaching someone, say you're teaching someone math, right? It's good for you to have learned at least one layer of math more than the level you're about to try to teach this person. And in a similar way, for this sort of training and coaching and training yourself, whatever, knowing a little bit deeper on why these things help can help you get a better sense of the big picture and figure out where you're going or if something confuses you or is... confusing or you don't quite understand. If you learn that sort of next level, that slightly one layer deeper, it can help a lot. I mean, this doesn't, there's two within reason, right? Like you don't necessarily have to go out and get like a exercise phys PhD to be able to write up some, you know, effective training plans for yourself, but sort of thinking along that way, thinking about the bigger picture and sort of the long term there can be really helpful. Yeah, totally agreed. And so, So starting to dig into this, everything kind of seems to point at PGC-1 alpha, or that's the typical culprit in terms of the main molecular signal integration point, right? All roads lead to Rome, I guess we could say. So this is like every signaling pathway that we've talked about. We've talked about calcium, we've talked about redox, we've talked about General Cellular Stress, Potentially Low Carb Stuff, AMPK, et cetera, et cetera, et cetera. So maybe we should think about also what are we doing with all of this training? What's improving in our muscles? Because PGC-1 Alpha, its mechanisms is going to help us understand what's going on here. So mitochondria has many functions. The main one we've been concerned about is the ability to maintain cellular homeostasis in terms of its energy state. How much ATP do we have and how much work can it do? So Wattstock number 40 has a lot more details on this in terms of oxidative phosphorylation and all that kind of stuff. What we really want to do with all this training is better maintain the cell's energy state. That is really the kind of main gist, the focus of the adaptation that we're making in the cell, which means just providing more ATP with the electron transport chain. And then we have, you know, temporal and spatial considerations to go with this. Also, again, in OneStock40. But on your bike... What does this mean? How do we feel this adaptation? How do we feel fatigue, resistance, and endurance? Kyle, do you have a good example of this? Because I was thinking about my first couple races. I actually remember my very first race, I got beat by a 10-year-old. Maybe it was like 12, something like that. And I remember getting to the... Yeah, typical stuff, getting beat by a junior. where I got to the end of the race and I was so gassed I couldn't even stand up to sprint. And I was looking at everybody else doing it and I'm like, how the hell do they do that? Then a couple races later I could actually stand up and sprint. I was like, oh, I've got a little more endurance. So like, that's my example. Do you have another good one for this? Yeah, I would say a similar thing with that where I remember, God, like my kryptonite as a crit racer was hilly crits when you're like, how can these people just keep you know they're the laps are sort of less than a mile and so every every lap you hit like a 60 70 80 foot hill and you're like this actually is a lot um a lot of climbing and I think that was shout out to the Westminster crit from 2013 that was always my my sort of kryptonite we're like wow like these people really can just when you're newer you're like you know I kind of have to go hard to stick with the group going up this hill but these people just like Crest the top and keep rolling. And, you know, I'm barely recovered by the time we get back down to the bottom of the hill, you know, just a few minutes later because it's a crit. And so you're like, man, just eventually you crack and blow up and, you know, that's the end of your day. Yeah. And so, you know, training is a way to improve those kinds of things. And so actually, I remember one of the road races you did, which was basically a hilly crit. I remember I was standing in the feed zone. You were off the front. You came through the feed zone. I was like, I kind of looked at you like, wait, what's going on? Is that Kyle? And you looked at me and you were like, I have no idea why I'm out here. Yeah, yeah. That was actually a lot of fun. At least those hills were like farther apart. You know, they were certainly like the 30, 40, 60 second hills. So you can kind of stand up and roll over. But you got more than a minute and a half of rest in between. Yeah, so that's the kind of stuff that we're talking about. And we're also talking about, you know, fatigue resistance that we can measure in terms of like, Does your five-minute power, one-minute power, your sprint power decrease as you race further and further and further? And that should improve as your fatigue resistance and your endurance improves. And a lot of this has to do with these signals, which signal for, well, all these signals are really, they tell the cell, part of them, well, they tell the cell that the cell has a specific need for more mitochondria and endurance capacity. basically is what a lot of these signals talk about. And some of them though are just signs that we are exercising. So like the falling energy state, AMPK, this translates, oh, we have falling energy state, we need more mitochondria. Calcium, presence of calcium, a lot of contractions for a long time. That's another way to say, hey, we are exercising for a while. Same with redox demand and like general cellular stress. So these things are all critical for the cell. Energy state, ATP's ability to do work, like I said, redox demand, how much, how many... You know, reducing equivalents do we have for the electron transport chain used to maintain the pH gradient in the mitochondria to drive ATP synthesis? Cellular stress, you know, MAPK, P38 is involved, but, you know, PGC-1-alpha is involved there too as like a, well, this is something cool we didn't talk about, but it seems like, you know, P38, MAPK Activation Stabilizes PGC-1 Alpha as a protein. And we're going to talk more about that later because I'm super excited. I think that's so cool. Calcium muscles contracting, yada, yada. And we have actually a couple more signals that we didn't really touch on, but I felt like those were all the big ones to think about because all the other ones, like, sure, they play a part, but they're not part of the big pictures of the signals that we can really consider as yada, yada. So we'll call them guest appearances. Like these are like a it's like a cameo from Spike Lee or not Spike Lee. You know, who's the guy from all the superhero movies like Spider-Man? Stanley. Stanley. Thank you. Spike Lee. Spike Lee is going to send somebody to my house to like punch me in the face. Spike Lee was in those Marvel movies. Come on. Yeah. So. So our cameos are from cyclic AMP and cyclic GMP. And these signals come from adrenaline slash epinephrine. And cyclic GMP comes as an internal cell translation of nitric oxide outside the cell for blood flow. And so these kind of have an impact on PGCO and alpha, but I didn't want to get too deep into them because we're pretty deep in the weeds already. But we'll talk about them briefly a little bit later. But the list we just made are things that, again, they tell the muscle a couple things. First, undergoing sustained contractions and energy use. This is a big one. Second, if you're training right, you are telling the muscle that its homeostasis is disturbed and it really needs to shape up. And this leads to adaptations. So it's like scared straight, but it's scared straight, but it works. Maybe a reference that some of the younger audience doesn't understand, but you know. Oh yeah. Listen to that Tom Segura bit on that. So what, and this is part of what, you know, what adaptation isn't. We will, we have yet to discuss this in terms of the relationship to burning carbs and fats. And we won't be discussing that because it's not an adaptive signal. And just to rehash real quick, adaptive signals don't rely on substrate use because substrate availability is varied. And by diet or any number of things like glycogen stores, things like that. So after a certain point, carbs and fats are basically equivalent and the body doesn't really care about where they come from or the cell doesn't specifically. And so I know this runs kind of counter to a lot of media out there at the moment, and I apologize if it makes me sound like a jerk kind of harping on that, but I think it's an important distinction to consider because I know there's a lot of people who think that lactate should be a signaling molecule, and it is far, far, far from being proven to be a signaling molecule. So anyway. Before I dig that hole any deeper, let's consider what is PGC1-alpha at all. And Kyle, I think you and I discussed this a little bit previously on like the calcium episode, but PGC1-alpha is a protein that's constitutively made in your cells. And it's part of a super family of proteins that are called nuclear receptors. So what a nuclear receptor does, it's a protein that takes signals of various kinds, like calcium and AMPK and all that stuff. And it turns them into gene transcription. And this is part of what are called signaling cascades or signaling pathways in cells. And like all of the ones listed above, it can take CAMK activation or AMPK activation and turn it into more mitochondria. It's like it kind of translates or transduces the signal into something else. Does that make sense, Kyle? Are those the right verbs you would say? Yeah, and I think that's, for people who've maybe taken college biology and things like that, you learn that that's, people think protein in your body is just like, oh, it makes muscles and maybe it makes parts of my ligaments and tendons, but actually your body is full of proteins that do things like this, like actually signaling and perform other functions. It's not just, it's not just for show, maybe? Yeah, if like, if you're, if I don't think I've ever met somebody who thinks this, but in case, but this would be like an extreme version of somebody thinking, oh, I shouldn't eat protein because I don't want to build muscle. It's like, well, you're riding a bike, believe me, you're not building muscle riding a bike. Yeah. Well, I think, too, we're like, maybe more of that is thinking like, oh, you know, why does my daily, why does the you know FDA even recommend I have to me a sedentary person get 50 grams of protein a day surely I'm not working out I'm mostly sedentary you're like oh well your body needs protein to do other things not just because you exercise yeah and your proteins break down at a certain rate like just based on entropy even if your body wants to keep them around they are going to break down um so so um where was I oh yeah so basically So gene transcription and turning these signals into adaptation alters what we could call your phenotype, the expression of your genes. Like my phenotype as a sprinter is very different from the phenotype of like Jonas Vingigo. Very different. He's got a lot more mitochondria. I have like two and they're working very hard. They're buddies with both of my brain cells. So these phenotypes, your changing phenotype alters to better handle the pressures that your exercise signals are sending. And, you know, for instance, sustained redox demand signals like via sirtuins. Like, they say, hey, it's a good idea, hint, hint, to make more mitochondria, we gotta spread out this load and service the more area of the muscle better, et cetera, et cetera. And so, sirtuins go up to their old buddy, PGC1-alpha, and pass on the good word, hey, more mitochondria are more better. Like, get to the nucleus, pass it on. And so, it goes to the nucleus, and PGC1-alpha's like, hey, okay, let's start doing some gene transcription. And nucleus is like, you got it, chief. And so, that's... kind of where we're going with all of this. So does that make sense so far? Because we really should, I actually kind of want to talk about the name of PGC-1 Alpha. Yeah. People probably, there are so many acronyms, I feel like, in bio that like, you know, people harp on like physics and astronomy for weird names and weird acronyms, but man. Bio and Biochem, just so many long names ended up getting shortened in weird ways and then people just throw them around like you, you know. Yeah, true. And also you don't have that many things to discover in physics. Like there are so many genes and so many organisms. It's like the number is infinite and we've got genes like Sonic Hedgehog and like Reaper and Death, which are awesome. And that's one of the reasons that science is cool. So anyway, what's in a name? Why is it PGC1 Alpha? And this has driven me nuts. forever until, you know, a couple years ago I learned this and I went, oh, okay. So why is it called peroxisome proliferator activator receptor gamma coactivator 1-alpha? That's a lot more, why isn't it PPARGC1-alpha? Well, the reason is because it's a member of the PPAR family. Like PPAR gamma coactivator 1-alpha, PPARP gamma G coactivator C 1-alpha. There you go. So it's PPAR, Gamma 1, Gamma Coactivator 1 Alpha. Of course. I know. So, but it's cool that it's a PPAR because PPAR family of proteins are what are called transcription factors. And so they're not active all the time. They respond to signals. Like when a sirtuin comes up and gives it a nudge about more mitochondria are more gooder. Yes, sir. Yeah. And so like, so for instance, like in, like in brown fat cells, this is actually one of the places that PGC-1 alpha was discovered in terms of its role in making more mitochondria and aerobic adaptation kind of signaling. Because, well, first of all, no, that doesn't mean ice baths are going to make you faster. Don't. I looked up papers on this, like when I learned, oh, this is where it was discovered. Okay, cool. And I immediately went, okay, endurance exercise, PGCE-1 alpha cold. And so, yes, it turns out that in completely untrained people, there's a little bit of a benefit. But in well-trained people, There's probably no benefit, not even close. I mean, when was the last time somebody was like, oh man, I got so much faster because I was riding outside in the cold all the time? Said nobody ever. It's always in the summer when people are like, man, I'm so much faster, I'm riding all the time, it's so nice out. That's when the things come. So you're saying that all those years I spent swimming in a cold pool, the coldness didn't help? How's your endurance right now? Well, it was a lot better back then. I was swimming 20 hours a week, but you know, shh. There you go. That's definitely not a violation of NCAA rules. So, well, actually, so I think it's interesting. Like, why does cold mean we need more mitochondria? Because what happens is to generate heat in our cells, we actually uncouple the proton gradient from ATP generation. We have separate pores that basically open and slowly release. It's sort of like if you've got a balloon full of air, but you need a piece of tape to do this. You put a piece of masking tape or scotch tape or something on the balloon, and you poke it with a pinhole. You can actually make a pinhole in the balloon without popping the balloon. And you put your finger over that. And so let's say the main nozzle of the balloon. generates ATP, this other one just releases air for no reason. Because like the main nozzle, you can go, right? And it's funny, but the other one just goes, and it's not nearly as fun. So that's what an uncoupling protein does. And our process of regenerating the proton gradient is actually relatively inefficient, right? Because that's why like total, we are what, 20%-ish efficient at turning turning like energy into work and the other 80% loses, we lose as heat. So everything's active except the ATP pump. Like how cool is that? That is cool. Yeah. So why is it also called a peroxisome proliferator? Because the original discovery in Xenopus frogs, it was shown to increase the number of peroxisomes. Peroxisome proliferator. All right. More on that in a sec. But let's talk about transcription and stuff because, right, this is a transcription factor. And there are a couple things, if you remember high school bio now, DNA, you transcribe DNA into an mRNA, and then you translate your mRNA by ribosomes into a protein. And there's a couple additional arrows that we can ignore, but this is known as the central dogma of molecular biology. and this is what Watson and Crick and all them were trying to kind of elucidate way back in the day and I and well they did a damn good job obviously so I remember that uh the um was it also the you may remember the the directionality that you can only transcribe in one direction like five prime three prime oh yeah yeah yeah yeah I remember that one so Yeah, fun times in high school bio. Yeah, well, for me, that was also college bio. And then we went, though, that was also like day two of college bio. There was a lot more on top of that. But anyway, so the first step of turning double-stranded DNA into single-stranded mRNA, because we're going to talk about mRNA quite a bit today, that's what PGC1-alpha has a hand in. And it doesn't have like a hand-hand, as in like PGC1-alpha gets in there and actually transcribes things. plugs into the things that do. It's like a really, it's like a turbocharger or something like that. I guess we could think about it. So yeah, we could talk about mRNAs in a little bit. So that's where the C in PGC-1 Alpha comes from. It's a co-activator. So like I said, PGC-1 Alpha doesn't touch the DNA itself, but it interacts with the things that do touch the DNA. And, yeah, like, I guess it just gets in there and smashes the throttle. Is that a good way to put it? Because the transcription is kind of happening, like, when you're not activating PGC-1 Alpha so much, like, there's a basal level of transcription, but when it gets in there, it's like, you know, you better limber up and put on your running shoes because this thing's going to just sort of go charging, charging down the DNA track. 

So there are a few different categories of transcription factors that PGC-1 alpha gets on. And this is probably, in terms of turning general aerobic signals into adaptation in the muscle, this is probably one of the reasons that, like I've said for a long time, and a lot of people have said this, where there's really only one Aerobic Adaptation Program, quote unquote, that we can turn on. It's that we are either improving the cell's ability to say it's energy state or it's not. And so PGC-1 Alpha kind of acts as a bottleneck. All the signals integrate to PGC-1 Alpha or something like that. We'll talk about the details of that in a second. But then PGC-1 Alpha interacts with certain other Transcription Factors that do things like transcribe nuclear and mitochondrial response factors to duplicate mitochondrial genome because mitochondria have their own gene sets. And this helps increase mitochondrial density. It also increases what are called peroxisome proliferator activated proteins, right? So these are our old peroxisome proliferator set. And these transcribe genes related to like fatty acid transport and oxidation and thermogenesis, like uncoupling proteins. So we can be nice and inefficient with our metabolism so we can generate heat. It also acts on estrogen-related receptor alpha. And this increases blood vessel formation around the muscle. Increase the expression of proteins that transport and store things for like glucose and fats. So like glucose uptake, gluconeogenesis, making new glycogen, lipogenesis, et cetera, et cetera, because fats have their own little bits of lipid droplets so that they don't have to go all the way from the fat cells too. So this is all stuff that... One Bottleneck, PGC-1 Alpha, turns on. It turns on sets of genes. It doesn't turn on, like, one gene. It turns on an entire, like, a giant subroutine of, like, we need to express all these genes, all these proteins, all at once. It's a very general program. It's interesting, too, because I feel like this is a completely side note, but, like, you know, I think... For a lot of reasons, testosterone is talked about a lot in exercise and sports and doping-related things, but it's not like estrogen is without its purpose in both keeping you healthy and then also exercise and adapting and performing well, like you mentioned here. It's not like, oh, you would be somehow, I don't know, even More Betterer as like a man if you had no estrogen. I don't know. Oh, yeah. No, totally. Like estrogen has, you know, protects like bone health, like cardiovascular health, if I'm not mistaken. Like it's a big deal. And like, and that's why, you know, like... like low energy availability and stuff like that. Like if, if women start missing their periods, like, like, and you're not making any estrogen, like this is very, very damaging to your health in the, in the longterm and even in the medium and short term. Um, and so that's one of the reasons that that kind of stuff is so bad for you. But, um, anyway, so the, um, so like a lot of papers that discuss PGC1-alpha and how, and how it kind of targets genes, uh, it, They all talk about what are called transcriptional networks. And I think that's a good way to consider the effect of PGC-1 Alpha because this is a way that PGC-1 Alpha takes a lot of signals, integrates them, and then turns them on to a giant spread of other stuff. Because could you imagine if like, like sirtuins and CAMK and AMPK and P38MAPK and all these things had like had to go in there individually? That would be chaos. Like, you couldn't physically fit that many transcription, like, co-activators onto, like, the actual transcription factors that express all these genes. It's, I, it'd be like, like, remember that Dane Cook joke where he was talking about somebody said that they called it, like, a thousand firefighters? So they're like, can you imagine a thousand firefighters? They're all bumping into each other and, like, going, hey, guys, like, what are we all doing here? Like, I can't work. Like, who called a thousand of us? Yeah, and I think, too, like, your cells have, as much as sometimes it seems like a cell is just like this watery bag of stuff, there has to be some sort of order. There is a lot of order, for sure. As much as it is a watery bag of... Oh, I've seen... papers that try to, that actually mimic the density and like the number of stuff in a cell with like biologically inert things and then put proteins and substrates and stuff in there and call that biological conditions and realistically it's like not even close because the cells are indeed highly ordered. So and speaking of order, how does active... How does PGC-1 Alpha get ordered to do its job? Because we talked about its signals that it integrates in terms of exercise and all that stuff. And we talked about just now its job as a transcription co-activator. So how does it get told to do work? Because with AMPK, it's like, we add a phosphate, then it gets to work. Cool. I mean, the regulation is a little more complex than that. That's the basic gist of it. So with PGC1-alpha, and this is glossing over, by the way, again, this is not as technical as it could get because that would bore me and also all of you, but adding negative charge to PGC1-alpha seems to increase its activation and its ability to be transported into the nutrius. So like proteins involved in our other signal pathways like AMPK, P38MAPK, sirtuins, Krebs, cyclic response element binding protein, All these things remove or add chemicals to the PGC-1 alpha protein. And like when a phosphate gets added, this phosphate has a minus one charge. So we are reducing the charge of PGC-1 alpha and that seems to enhance its ability to be imported into the nucleus. And so the more negative the charge on PGC-1 alpha means it's more likely to go do its job. And that's step one. And step two, is that the negative charge in the protein allows it to better interact with the DNA-bound transcription factors. And this seems to happen through a lot of hydrogen bonding. And hydrogen bonding, as Kyle, as you know, is a lot of kind of electra-weak, well, that's, sorry, that's a physics term. But, well, you know what I mean. How would we describe hydrogen bonding? Weak magnet attraction? Yeah, I think that, Biggest thing is like there are okay so there are several different ways that that atoms and molecules can decide to form to decide to form a bond and hydrogen bond is when you have like an atom that is particularly negative negative charge on some area yeah particularly negative charge and this doesn't have to be like it is an ion this is more like because Certain nuclei can have a more negative or more positive. You can cause like a sort of gradient from more positive side of a molecule to more negative side of the molecule. And then we have those more positive and more negative sides. You can get those sides to actually want to link together with other things because opposites attract. Yeah, and what would we call oxygen? An electron hog, an electron slut, an electron... And so it's like hogging electron cloud from the hydrogens, and that's like the original kind of thing of hydrogen bonding, is just hydrogens being like, no, no, no, no, you take it, you take it, we're good here, we're good here, we'll be a little positive, it's cool. Then those hydrogens find another, like, oxygen on another water molecule, and they just kind of go and stick together, but they can be pulled apart pretty easy. The force binding that isn't that strong. So there's more that can be done with PGC1-alpha than adding a phosphate group. Like most of the papers I read list six potential modifications that can be made. A lot of them aren't understood what their function is. But one of them is actually really, really, really cool. So we should probably talk about, before we talk more about that cool stuff, Does an increase in PGC-1-alpha protein have a positive effect on endurance adaptations? Because one of the things that we see and one of the things that I'm sure a lot of our listeners are thinking about right now is, okay, if we've got this protein and we activate it and make more of the good stuff in the nucleus once we're exercising, if we have more of this protein, How is that something that can potentially translate into aerobic signals? And some papers would say yes, and some papers would say no. So let's... It seems like one of those things like maybe I'm the only one who's seen a lot of this, but there are those companies out there that will sell you like... Bottled ATP and a little dropper in it and stuff like that with the idea that you could, oh, if you just, oh, if ATP really is the, you know, the driving force behind cellular metabolism, sports, things like that, maybe if you just drink or dropper on your tongue a little more ATP before you train or you compete, it'll help somehow. That might just, I don't get those, but I get a lot of other weird stuff advertised. This makes me think of that, like someone being like, I know, what if we just, what if we just bottled PG-C1 Alpha? Yeah, well, you know what else has ATP in it? Literally every piece of organic matter that you eat. Meat, fruit, vegetables, grains, take your pick. But, okay, sorry, I don't want to. I'll take a cut of that ATP potion profits. No, I'm just kidding. No, I won't. So one of the things about the studies on PGC-1-alpha is that it gets made at super physiologic levels in order to show its effect. And the same thing happens with other things. Like if we make a constitutively active form of AMPK, for instance, that can have an outsized effect because In reality, AMPK has never activated that much for that long, you know, in perpetuity in a cell. And it's a way to exaggerate something. It's sort of like, Kyle, this is one for you. It's like, remember your first ever physics class and somebody's like, imagine a cow as a perfect sphere on a frictionless surface. Yeah. So that's kind of the equivalent of these studies. PGC-1 Alpha in reality has a very short half-life. And I believe, I don't know if I have it in my notes here, but I think I remember seeing it as about two and a half hours. So it has to be in your water bottles then. Well, and I mean, believe me, there is so much study on what makes PGC-1 Alpha transcribed. And based on that, if you... you know look at the super physiologic level papers like you might think okay yeah if I can make a lot more of it this might have a beneficial effect but at the same time it's being what's called ubiquinated quite frequently I think it's being ubiquinated it's got a pathway of degradation where if it's not being used it kind of goes away and actually we're going to talk about ubiquination ubiquitination in our next Wostok episode which is going to be I'm very excited for that one. I'm excited for all of these. Whatever. You guys know me by now. So the signals that coincide with exercise have a pretty strong positive effect in higher transcription of PGC-1 alpha itself, like calcium and epinephrine. You know, epinephrine yields cyclic AMP. But they're two very strong general kind of we are exercising signals that have a big effect in helping more PGC-1 Alpha get transcribed. But Evolution decided this is probably a good idea because... The use of the proteins in transcription probably has some effect in degrading them based on their use and there's the kind of entropy in the cell. And so if you are exercising a lot, you may end up with most of your PCC1-alpha in use and now you're shorthanded. Now what do you do? Especially if you've got a lot of mitochondria there and a lot of cells, a lot of muscle mass or a lot of muscle nuclei. Yeah, you probably are going to be shorthanded. Like, now you've got two firefighters instead of a thousand. What do you do? You want something in between, somewhere in between. Yeah. So it seems to me that we're still missing a couple key pieces of evidence in determining... if more general PGC-1 alpha transcription actually has an effect on performance. And this is going to be one of the big points of the episode, by the way, is your performance. This is a big thing about all of these Wastock episodes, of course. So more gene transcription, next step, more protein made and protein integrated into the cell. Next step, better performance, question mark? Like question mark on all of those things. But that's the theory. But the magnitude matters too because if one workout, for instance, has 10% more PGC1-alpha mRNA transcription than workout two, you may not see detectable level of performance with an improvement like that because there are so many steps. You make more PGC1-alpha mRNA. But how much of that gets translated? How much of those get used? How strong is the signal to tell the PCC1 Alpha to get used? And this is also something that would compound, if it has an effect, it would compound over subsequent workouts. But then again, if you really overdo it, now you're overtraining. So you don't want to big brain this stuff either. So now, for instance, if we see these workouts done, like workout one and workout two, if they're done for eight weeks, I doubt we're going to see a 10% performance improvement in the one with 10% more PGC-1 alpha mRNA transcription. Because this is a thing that a lot of studies look at. And I've wanted to include a lot of studies on the podcast that have this as their measure of effectiveness. And I throw them out. I'm like, oh, well, because... I cannot in good conscience say this is going to have a positive effect because we don't see a performance improvement and that's something that I think a lot more studies need to use but at the same time if you are working with like myotubules which are just little cultured muscle cells like what performance are you going to get out of these things like they can't run they're in they're in water for instance like they're they're just they're yeah they're literally in a dish like yeah talk about like petri dish like they're sitting like Yeah, so like, so you may need to see double the PGC-1 alpha or whatever mRNA transcription to see a 1% detectable difference. Now that would be a result. That would be something I'd be very interested in, you know? So, excuse me, but I've also yet to see any of this done with like single fibers. I bet there are some papers out there with single fibers. I personally have not seen them. If you have seen them, please send them to me. And so a lot of this is actually whole-level muscle, like homogenate. You take a muscle sample, you grind it up, like literally in a blender, like you're making a margarita, and then you kind of figure out, okay, here's how much PTC1-alpha we have, right? Muscle margaritas. And so, for instance, if we have something that has higher intensity, like higher motor unit recruitment, I would think we should have a little more mRNA synthesis because we're getting into larger motor units. But of course, the more well-trained motor units down at the bottom may not have any. So this is something that needs to be kind of sussed out in the literature. We need a lot more experiments, in my opinion. So it's also my opinion that given what we've discussed in previous episodes about the combination of duration and activation level, so Kyle, your area under the curve, of PGC1-alpha activation. I think, to me, that is what matters more than the absolute level of protein or mRNA transcription. Do you think I'm way off base? Please be honest if you think I am. No, I think that makes sense, but it's kind of like the thing where, yeah, if you would... When you say that sort of area under the curve, like total integrated intensity and volume is what matters the most, that probably mimics the experience of most people. Like you could imagine that there isn't really a way to shortcut some of these things as much as people would want there to be. And just, oh, if you could, oh, if somehow we could figure out just one way to trick our bodies into, you know, doubling. the amount of transcription. Oh, well then we'll double the effectiveness for the same amount of time. Like, it'd be cool, but yeah. And I think some of this too is also, it's not the only, like there's not, there's rarely ever some silver bullet magic one factor that if, oh, if you just change this part of your workout or magically Do this one thing, it will, this is the key driver behind all, all of it, um, which would be cool if it was, but unfortunately, aerobic adaptations that are much more complex, where there isn't just this one, uh, you know, one marker you have to look at to determine exercise effectiveness. That would be cool if there was one thing you could easily test for in your blood that would tell you at an absolute sense whether or not a workout is effective or not. That would be really, really cool. Yeah. We don't have that. We can't even tell if somebody's, like, tired and overtraining with a blood test at this point. Like, we're not even close to getting a blood marker for aerobic adaptation. But, you know, it's... Actually, it's funny, this is not in the notes, so forgive me, I might end up cutting this, but I remember reading a paper that was published in like 2015 or 16 or something like that, which tried to look at all of the ways that AMP, yeah, like, it was like AMPK and calcium and all, like all the signals that PGC-1 alpha integrates. Not only that, but also looks at the conditions under which PGC-1 Alpha is transcribed. And it tried to make some recommendations about how to do, how to improve this kind of stuff. And I use that as kind of the basis for trying some of these protocols of like low carb training and things like that and facet training, which I did not ever have any of my clients do. I did that myself and it fucking sucked. And so we tried to do all that kind of stuff, or I tried to do all that kind of stuff, on myself or with some willing participants, like I've mentioned on the podcast before, and none of them really worked. None of them really worked that well. And in a lot of ways, I think we would have had a lot better results if we had done the same training fully fueled up. And just because of all the stuff we've talked about, because your muscles know the difference, like we talked about in the AMPK episode with altitude. like if you're up at altitude and you are like your FTP is normally 200 watts or 250 watts let's say and at altitude you're at 200 watts the AMPK activation in your muscles riding at FTP are lower because your muscles know the difference so that's one of the things about being under fueled is the quality of the workouts makes a difference and when I read this paper initially and a lot of people had you know we were all kind of excited about it like oh cool this guy knows what he's talking about then we tried to put it into practice and then we all kind of agreed at one point it was like this is probably something for like soccer players and football players and probably not dedicated endurance athletes because this is fucked. I remember doing one fasted ride and I brought a banana in case of emergency and oh my God, did I need that banana and then I crawled home. It sucked. Yeah, and you could imagine too, yeah, if you could somehow have the same workout quality while low carb or Fasted or under-fueled or whatever, then hypothetically there's, you know, that's a different experience. But yeah, if the under-fueling is just hampering workout quality, you know, why are you even there if you're not? Like, you may as well just nap. Yeah, precisely. Yeah, so, and that's going to be one of our kind of ultimate points that we're going to get at. But anyway, so my final thought on what we were talking about is that PGC-1 Alpha is very inducible, let's say, by various conditions in muscle cell and also in other tissues. And we're going to see a lot about that right now because I wanted to look at a couple experiments on PGC-1 Alpha knockout and see just how critical it is. There are some very surprising results. And that's one of the reasons I've been so excited to get to this because I heard about these experiments a while ago and I was like, I gotta get to these in the podcast one of these days. And this is that day, I'm super stoked. So in the meantime, I, in the show notes at empiricalcycling.com under podcast episodes, I'm going to link three really excellent reviews on PGC-1 Alpha and how it works because we've glossed over a lot of it. But if you are really interested in this, I highly recommend going to read these. I'm going to link them. I believe two of the three, if not all of them, are free text. So you don't have to, you know, Sci-Hub it or go behind a paywall or anything like that. 

Looking at these studies, I actually wasn't sure initially if we should go with a gain-of-function study because a lot of the stuff we've looked at so far has been both like gain-of-function, so we're going to make a constitutively active version of, I forget which ones we've done, but then we've also looked at what happens if we block these effects. Like I think in the calcium episode, we looked at caffeine has an effect that would make myotubes contract and the substance called dantrolene would block that effect because it would out-compete the receptor and so caffeine had no effect. And so that was one of the ways that experiments work in order, like, okay, we're gonna... have this thing, but we're also going to like block this thing so we know for sure it's this thing instead of having this thing and not having this thing because there might be something else going on. So if you block it very directly, you can absolutely say for certainty it's this thing. And I love that about a lot of these experiments. So we're going to go with loss of function and we're going to look at two papers that look at loss of function from basically the same group of researchers. So Kyle, Let's start with you. What do you think happens when we delete PGC-1 Alpha in mice or knock out PGC-1 Alpha as papers call it? Listeners, ask yourselves too. What happens? Are they born? If they live, can they adapt to exercise? Do they generate body temperature at all? What happens? I feel like it, I mean, I assume, I kind of would guess that they're born and then they have like, they're just like, Very low energy, I don't know, you don't have, you just feel like you're maybe tired, sloth-like all the time, like maybe they don't, you know, you think mice, you think they want to run a lot, right? Maybe they just don't, or they can't run the same volumes and intensities that sort of control mice can, and then, and then, and then. You may also wonder if they have poor recovery, if they have bad cellular metabolism or are missing this key signaling component of cellular metabolism or if they have weird premature death because of a lack of turnover and things like that in their cells. I'm not really sure. Yeah, actually, you actually sound like an article I read from One of those fitness scammer guys. Do you have low PTC1 alpha? Do you have low energy? Are you chronically inflamed? Is modern medicine letting you down? If you have low PTC1 alpha, co-exercise. That's it. So here's what happens. I've, of course, linked the article in the show notes. So researchers bred heterozygous mice. Meaning they have a functional copy and a non-functional copy of PGCO and alpha, or plus slash minus is the convention in gene studies. And I like this design personally because you get, when you breed these two mice together, you get a Punnett square, right? Now you get plus plus, plus minus, and minus minus mice in the exact same litters. So you can actually compare wild type plus plus, both have the gene, and knockout mice minus minus. And yes, they did live. And when they started observing their new mutant mouse babies, their first observation was that they had no unexpected deaths. Hmm. Yeah. But the knockout mice were 15 to 20% low on normal body weight. And they had some different body composition and relative muscle size things. Like, it was all kind of fine, more or less. So what they did was they looked at muscle biopsies of the soleus muscle. And the soleus is usually picked because it has typically a very large proportion of type I muscle fibers. It's a very highly active muscle in mice. And the other muscle, if you're curious, is the EDL, the extensual digitoris longum. And this one has a lot of fast-twitch fibers. And so when you usually look at these studies, they'll either... They'll usually harvest both of those and compare those two for, you know, fast versus slow twitch fiber comparisons. So the knockout mice had fewer and smaller mitochondria than in the wild type mice, but not absent. They had about, yeah, two thirds of the total mitochondrial volume of the wild type mice via histochemical staining, which is basically you take a Muscle Cross-Section, and you kind of take a look at what's under there. Or it might have been TEM microscopy instead. I forget. But so pretty cool observation, right? Yeah. Yeah, yeah. I mean, that's interesting that it's a measurable difference, but it seems like it wasn't one of these potentially debilitating knockout studies. But yeah. Yeah. So they also looked at other proteins like TFAM. And back in 2005, when this study was done, this was a known interactor with PGC1-alpha. And it turns out it's necessary for function with mitochondrial DNA and stuff like that. They also looked at cytochrome C of the electron transport chain and stuff like that. And pretty much everything across the board is about 30% to 50% of the normal amounts. of the knockout versus the wild type. So everything's down across the board. But the most surprising to me was the mitochondrial function test. And this is, by the way, if anybody's thinking mitochondrial function has something to do with lactate, usually in the literature, mitochondrial function is basically a mini VO2 max test. where you isolate mitochondria and you test them all at states 2, 3, and 4. Don't worry about that if you don't know what that is. 2, 3, and 4 mitochondrial respiration rate and they look at oxygen consumption per unit mass. So it's like picomoles of oxygen per microgram of mitochondria, something like that. It's the world's smallest VO2 max test. As you say, does that scale to milgrams or liter, like liters per minute? People have tried to scale that and it turns out that it doesn't really scale that like that. Oh, okay. But it basically means that for the same unit volume, the mitochondria are basically in terms of ATP generation and electron transport chain function, more or less the same. But the knockout mice were about 10% down at true max. You know, at the submax states, that the not quite VO2max stuff, they were basically identical. So we don't really have a true loss of function. We have a loss of volume and mitochondrial mass more as the thing here. So now these poor things start to exercise. So this is just like their preliminary observations. The knockout mice have a generally lower level of activity by about half. Makes sense. Yeah. And when given a run to exhaustion test, the wild type mice on average, and this is like a mini ramp test, they lasted about 600 seconds and knockout mice didn't even make it to like 100 seconds. 

Woof. That's not very much. Yeah. 100 seconds is... Yeah, but it's a ramp test, right? Even sprinters can... I know, right? So on their whole body VO2 Max, the VO2 Max was about 15% lower. So it's not precipitously lower, but it's definitely lower. Yeah. And then, because we know that mitochondria and uncoupling is involved in generating heat, they then expose these poor mice to the cold. They expose them to 4 Celsius for 5 hours while monitoring rectal body temperature. And while type mice core temp dropped by about 3 degrees Celsius total in a linear fashion, knockout mice lost 13 Celsius in their body temp. Oh God. And not only that, it was an accelerating downward arc. Like it was going faster and faster and faster the colder they got. Oh God. I know, poor mice. So it was- I mean, that's like hypothermia like immediately. Well, here's the cool thing. Uncoupling protein abundance doubled in both types of mice. So they still actually had an adaptive signal scent. and they were, you know, they were responding to the stimulus. They were just, like, they got lower mitochondria total, so they had a worse time at regulating their body temperature. Yeah. Yeah, it's like they, without that one signaling or messenger, it's like one stimulus comes in, but then there is no one to continue passing along the message, so to speak, that, you know, things are bad and we need some help here. Yeah, it's like, It's like, it's like, well, this is going to be mixing temperatures, but it's like if you've got a fire to put out, and if you've got a normal amount of mitochondria, you've got like 10,000 buckets of water to put out the fire, and that's like you generating body heat. So sorry to mix the temperatures, but if you've got PGC-1 alpha knocked out, you apparently have like three buckets of water. Yeah. And they're all friends. Oh shit, oh shit, oh shit, yeah. And they're all friends with both of my brain cells. This also happens especially in brown fat because it's mitochondria, by the way, that makes the fat brown. Like this is your visceral fat. And this has, of course, visceral fat being a good idea to keep your organs warm, for instance. And so there's a lot more stuff in this paper that we're just going to summarize. So these rats have problems in the liver. By the way, don't click on the paper and go read it if you're squeamish. They have photos of the dissections. They have problems in the central nervous system like lesions. You don't want, you don't want nerve lesions, generally speaking, it's probably a bad way to go. Stay away from nerve lesions, spina bifida, the usual stuff. And so, and it's not in this paper, but we're going to toss the kidneys into the ring too. Eyes are ATP hogs. Look at Elhan Libris Hereditary Optic Neuropathy. That's a tough one. And that's like loss of one of the... Electron Transport Chain Complexes, I think it's number one. So a lot of vision problems in those poor people. So we can go down the list, but wherever tissue is highly metabolically active or energetically intensive, if you have a PGCO and alpha deletion, you are going to have issues. The cool thing was they had no insulin resistance on a high-fat diet, these mutant mice. They had better glucose tolerance. That's cool. So far, our list of organs highly impacted by the loss of PGC-1 alpha, skeletal muscle. A lot of them. Brown fat, liver, nerves. And just from my reading of the literature preparing for this podcast, I'm also going to toss in kidneys, skin, veins and arteries, white adipose tissue, white fat, bone marrow, and everything. Just everything. Everything is on the list. Everything. Woof. Yeah, that seems bad. Yeah. But... The mice didn't die. Silver linings, I guess. And, you know, because that made me wonder something, and I could not leave it there. Because, you know, they didn't die. The mitochondria is obviously very important to survive as an organism. And is there more going on with mitochondrial expression? Are there backdoor pathways? Are there other things going on? Are there things that serve the same function as PGC1-alpha? that they didn't know to look for. So now we've got one more paper by the same group. They followed up in the study. And no, they didn't. Sorry. This is a different group. Because as soon as I started looking for this, I found two papers by the same group. And we're going to talk about these both in brief. This group did something a little bit different. And I think actually, I don't have this in the show notes, but they did what's called floxing. They floxed the PGC1-alpha gene. And so this is a really, really, really, really cool way to knock out a gene because previously, 2005, you knock out a gene, you breed it, and the gene is absent in all tissue. So when you flox a gene, you can actually have temporal control. You can say, I want to delete this gene at like three months of age for this mouse instead of like having it gone from birth. And you also get to pick the tissue. Interesting. Yeah. So that's how they knocked out PTC1-alpha in this. So I love that. And they left the rest of the body alone except skeletal muscle. So what they did was they had the two groups of mice, as usual, and they let the mice run at will for 12 days. And what they saw was no reduction in total exercise for the knockout mice, but they had a small and non-significant reduction in running speed. That's it. And take a wild guess. Do you think that the knockout mice got faster and had better endurance, for instance? Possibly? I'm going to guess no. I thought so too, and we're both wrong. Huh, okay. Yeah, the running speed increased the same for both groups over the 12 days of the running at night, and they ran for, I don't know, I think that, I don't have this written down, but I think they ran for like 8 or 10 hours a day or something like that. because they're mice and they just do that. Yeah, yeah, yeah. Yes, but so like remember, whole body PGC went off a knockout, made you very lethargic or made the mice very lethargic. But these mice, just in skeletal muscle, not so lethargic. I think that's pretty cool. That's super interesting. Yeah. And a little, yeah. A little unexpected, yeah. Very counterintuitive, yeah. Yeah. So they started looking at gene expression. right after a bout of exercise. Like, what happens? What doesn't happen? So PGCO and alpha expression was basically absent in the knockout mice as expected. And if you look at their Western blots, you see tiny little lines. This is probably like contamination from other tissues, so not a huge deal. What they saw was expression of like mitochondrial, you know, things that you need for electron transport chain like cytochrome C oxidase as in electron transport chain complex four. The mRNA expression was similar for both. And it turns out most gene expression was nearly identical. And it turns out they didn't even see a relative increase in what's called PGC1 beta to compensate. And so the authors conclude As their title says, PGCO and Alpha is dispensable for exercise-induced mitochondrial biogenesis in skeletal muscle. Huh. Interesting. Yeah. So maybe it's not so important as we think. Maybe. Dispensable. I feel like that's a strong choice of words. Dispensable. It is. But they got knocked out and they got faster over time and didn't seem to be a huge deal. Weird. I think whole body knockout was probably a lot more impactful, but also it's because when an organism is deciding to exercise, there's a lot more factors than just, hey, are my muscles feeling okay? Right, right, right. It's the integration of all that stuff. Can you imagine too, like, we sort of take for granted, right? If you're healthy enough to exercise, you sort of take for granted that all these other systems in your body have to be working pretty well. for you to have a good time doing it, you know? Yeah, yeah. It's kind of just one of those things maybe, I mean, I don't even, it's not like we think about that when we go to the gym, like, oh man, I'm really glad that my eyes are functioning today or whatever, like, you know, my skin is... has normal cellular metabolism going on, so that's good. Skin is a real key component to accomplishing this workout. Yeah, you're not like, hey, how are my kidneys functioning? What's my glomerular filtration rate right now? Can I go to the gym? Yeah, nobody does that, but I mean, this is why, as a coach, I get concerned when people tell me that they're kind of lagging in terms of, like, motivation or just general energy. I'm like, oh, shit, this is an emergency, because I know once... Highly Motivated People Start Feeling Like This. I know, like, this is a giant red flag. But one of the things to think about now is this PGC1 beta, because one of the things that we've been, we've been, I have been deliberately leaving out is that there's more to this kind of gene transcription pathway than just PGC1 alpha. So PGC1 beta, and there's a third protein in there also that, third protein by the way is, we'll get to that. These are all extremely important in these signal pathways and they all seem to have somewhat the same functions. PGC1 beta and the third protein don't really, they're not nearly as studied obviously, but we're gonna talk about this. So in this study, this is a follow-up study from the one we just looked at and they deleted both. PGC-1 Alpha, and PGC-1 Beta, and ran nearly the same tests. And so now they start seeing some markers of decreased mitochondrial content in skeletal muscle, like less mRNAs, less electronic transport chain components, but not all of them, just some of them. They also saw a decreased TTE in the RAM test, like the knockout mice only went about 30% as long as the control group. So now we're starting to look like our whole body knockout mice. Yeah. And by the way, at this point, I wanted to point out that the researchers are now using female mice from this point on in the study. And I think they did this in the last study too, because they said the female mice seem to all do better in the exercise tests. So this is one of the rare things in science where it's almost entirely female experimental group, which is awesome. We hardly ever get these. So this time what they did was they made all the mice sit on their asses for 12 to 14 weeks. And then when both the wild type and knockout mice were at their least well-trained, their most couch potato, what's the mouse equivalent of a couch potato? I don't even know. I just don't have couches, right? They have little grassy piles. Yeah, hay. I haven't had a mouse pet. I have had hamsters. Pretty close. Just kidding. Not really. So what they did was they made the mice sit on their asses for 12 to 14 weeks, and then for both wild-type and knockout mice, They then started to exercise them and see if they started to get any improvement in their endurance. And it turns out, yes, they did. They saw the same two-fold increase in exercise performance in both wild-type and knockout mice. Interesting. But the knockout mice stayed at about the same 30% of the control group in terms of TTE, distance run, and work output. like the whole way across even as they got faster. So, right, another unexpected result. We're missing PGC-1 Alpha and PGC-1 Beta which may have been doing some of the heavy lifting. So, clearly, there is a big impact but clearly, even with them knocked out, these poor, completely untrained mice are still getting faster. So, this is pretty awesome. Despite having knocked out one of the things that we've all been talking about as like the thing for aerobic adaptation. And, you know, it's pretty big, but turns out it's not like the thing. There's more going on. So they also looked at how despite the knockouts, Electron Transport Chain mRNA Expression was greatly reduced, but the protein expression was different, but not across the board for all the proteins they looked at. So there's a decoupling between mRNA expression and protein expression. It's not like the same level of mRNA. translates into the same amount of protein. And they are seeing this very directly in this study. And this is one of the reasons that I harp on this all the time is because I see things like this occasionally in literature where I think this is not a one-to-one relationship. And this is part of what's informed all of my assumptions around being critical of these parts of the literature. Interesting. Yeah. So for their mRNA and electron transport chain protein expression, this is all like closer than just the 30% of the control group. Like they had some activity compromise, but not in all of it, of the electron transport chain complexes, like one through five, or I think one through four. I forget if they looked at five. So they saw the same relative increase in mitochondrial protein relative to the exercise dose. It's a, I think this is a really cool result because it's another way to hammer home the point that, you know, mRNA and protein expression and performance are, they have a very complicated relationship. We really cannot say they're linear, but also I still think it's cool because there's more going on to this with this than just like PTC1 alpha and PTC1 beta activation expression, et cetera, et cetera. Like, you know, we can, you can do all the fancy stuff you want, but. Even if you don't have this stuff, you can still do okay. So maybe probably the last interesting note here in terms of the experiments that they did was they looked at the deep quadricep muscle. So basically what happens in recruitment, this is what the paper said anyway. One of the few things I did not go and verify is that the deeper muscle is used more for postural stuff. Frequently Recruited, just walking around. And the larger motor units, and as you get outside the muscle, seem to be used less, only for the most intense exercise. And so what they did was they basically looked for, and I don't know if that's true or not, but they looked for a sample of the quadricep muscle about halfway in for the control and the knockout mice. and they looked at what's the mitochondrial density. And in the knockout mice, they did not see any increase in mitochondrial density from the exercise intervention, but they saw plenty of improvement in the wild type mice. And they still had performance increase, they still had more mitochondrial protein made, et cetera, et cetera. I've got a couple explanations for this. The authors went into a couple things, but I've got some thoughts too. So I thought that maybe the lower speed of the knockout mice running didn't recruit as much muscle mass if indeed it does fall into the same pattern. And so maybe they just missed the portion of the muscle like super deep in the quad that was being recruited. That's possible. I'm not entirely sure. Second. is that the increase in performance happened due to more mitochondrial protein content but not necessarily more visible area on the image. That to me is the most likely. And also kind of co-equally likely I think is that there's another compensatory mechanism for the improved cardiovascular fitness like more VO2 max, more glycogen storage or transporters or any number of these things that don't necessarily have to correlate directly with more mitochondrial mass. Yeah, it was interesting when you said that one population only lasted like 100 seconds. The first thing I thought of is like, they just- They did a kilo. Yeah, toughed it out on anaerobic capacity and I was like, nah, I'm good. Yeah, and if memory serves, and my memory for the papers I've read is pretty fuzzy at this point, there have been so many, but- I believe I've seen some papers where there's been knockouts and stuff like this or they've looked at people with congenital issues and there are indeed some compensatory mechanisms for stuff like this to make the muscular bioenergetics work in order for organism survival. So it wouldn't surprise me at all if something like this is happening. So, excuse me, to finish up the study, the authors conclude that There may be a third member of this protein family at work. So we've got PGC-1 alpha and beta. This one's called PGC-1 related coactivator or PRC. But here's the one where if we delete it, it's lethal from birth. So that's a big one. So PRC is potentially more important. for Organism Survival and Adaptation, but it's not terribly well studied that I'm aware of, and it doesn't seem to be as, I don't know, I just haven't seen a lot about it. But anyway, the authors also note that AKT, P53, P38, and there are potentially other unknown PGC1-alpha independent pathways that happen for aerobic adaptation. And I think All of this makes sense because you don't always get exercise-related signals to need mitochondria. One example would be how mitochondria and related bioenergetic genes and proteins are all tied up in the pathways for cell division and cancer. There are a lot of other things in the cell that can turn on the same sets of genes. And it's potentially true that they're getting activated and they just didn't test for them in this study because Well, first of all, there's a thousand things to test for. So that's kind of cool. I mean, it is interesting because you hope, just from a survivability evolution standpoint, you hope that your body does not have a lot of single point failures where like if this one thing happens, aside from like, you know, there are very obvious things like major organ failure and things like that, but you would hope that a process that may be subject to more It's not random chance is the wrong phrase, but say like transcription errors where you get your body just doesn't make this one protein quite right, right? If that, you know, obviously there are some genetic diseases where this happens and it is catastrophic and terrible, but for things like this where you need transcription to happen or if you make it, you hope that sometimes if the transcription goes wrong and you produce some proteins that don't really work the right way, that you're not completely left out to to dry, right, where it's not, yeah, a single point failure where you're totally screwed after that. Yeah, dry of mitochondria. Yeah, exactly, like, oh, this one, this one time you have this, and people, and just so people are aware, like, your body makes transcription errors all the time, you know, and it's, it's fine, you don't, you don't even notice. Yeah. And it's great because your body's figured out how to. Not figured it out, but evolved, not just completely break down if you have some transcription errors. Yeah, if you ever start looking into these, like for instance, like chemical homologues are in the way that mRNAs and DNA gets transcribed, like this is why that more than one codon transcribes for the same protein. Yeah. And a lot of these codons are chemically very similar. And so if a normal substitution happens, you are still going to get the same protein. So that's one of the cool mechanisms that you can get for protection. Yeah, for sure. And so then within this, it's like, okay, so if there are some of these cases where, like you said, there is one very unfortunate trial where they knock out something that is... a fatal defect. But generally speaking, you hope that your body has a way to get the majority of the signaling done it needs to to keep you alive in a way that is at least has one one layer of redundancy. Yeah, completely. And yeah, sorry, keep going. Oh, no, it makes it makes sense. Like, generally speaking, there's a lot of these things. It's both involved with exercise, which may not be that critical, but recovery from exercise and adaptation to exercise is the thing that you want your body to be able to do. Yeah, that is something that your body has figured out multiple ways for you to sort of at least recover, mildly recover from. But you never know. Yeah. I think in addition to that, kind of my general thoughts are that stuff like this really muddies the picture of aerobic adaptation and its relationship to performance especially. Because PGC-1 Alpha and PGC-1 Beta, sure, they seem to be absolutely necessary to expand the mitochondrial reticulum and all the other good stuff. And also, these mice didn't exercise for that long. These are all new gains that these mice are getting right now. We're not talking about the Tour de France winning mice here. Could I see Ratatouille, like with the Tour de France? I desperately need this movie now. I mean, Fogaccia does have that hair that sticks up, right? Which is like the key. Oh, he's got the mouse controlling him. Now we know. Yeah, exactly. Yeah. Yeah. Yeah. Take the helmet off. Show us Remy. Let's... So, I think there's... I think this really just shows that there's a lot more knowledge that needs to be gained about all this stuff. you know there's more to improve muscular endurance than just like PGC-1 alpha and PGC-1 beta activation and you know although clearly they are extremely necessary and I think if these mice had exercised for even longer we would have really seen some and you know people had looked at other stuff like you know capillary density and fiber type and all that stuff we would have seen some even more stark differences between the two but I think at least preliminarily it's you know It's a complex picture. All we're really trying to do is better maintain the energy state of the cell. And that's what all these adaptations are about. Again, there's one program switch for this stuff through our usual suspects of general exercise signals like calcium, energy state, redox demand, cellular stress, epinephrine, nitric oxide, like all of our old friends. And how we use and misuse this information is... Well, we're just going to do the rest of the podcast on this because we've got some great listener questions. But I think in my opinion, first of all, you know, we're going to go back to the same usual advice of ride more, progressively overload, make sure that you're recovering using your workouts as your guideposts for progress and to show how your recovery is going. Because like we said about the AMPK stuff, you know, Besides the cool science here, progressive overload and your muscles knowing the difference between a good day and a bad day, it may feel as hard, but et cetera, et cetera. So I have a funny aside. I was watching some training video from some bodybuilder on YouTube or whatever, and having to say like, oh, now I really realize that Last year, I paid really close attention to progressive overload and actually writing down sets and reps and weight and then, you know, trying to keep track of that and it worked really well and I was like, oh my god. Like, I get it that like a lot of people just go to the gym just to get a workout in, you know, or ride their bike just to get a recreational rider just to feel good, feel good about yourself, have fun, you know, ride and that's fine, nothing wrong with that. But if you're like a competitive bodybuilder, that's like your hobby of choice. And you're like, oh, I just figured out that last year that progressive overload seemed to really help. Oh, God. And I have those friends, too, who go into the gym and just grab the same weights and do the same sets and reps and everything. And some days they feel good, some days not so good. And I'm like, well, if you feel good, do more. And then if you're not recovered, then go home and then wait until you're recovered. It's, the number of things that, that endurance athletes can learn from, you know, the strength training world, because the strength training world, we've talked about this like 20 times on the podcast before. It's like, you can either lift the weight or you cannot. There's no in between. There's no like, oh, I kind of lifted it. I kind of rounded my back. It's like, well, you rounded your back. Like, that's not a lift. Like, you're going to hurt yourself like that. So it doesn't count, et cetera, et cetera. But anyway, back to the nerd stuff. exercise intensity in the signals and stuff like that. Because, you know, the activation of the signals, of all the signals that, you know, integrate into aerobic adaptation, they do typically increase with intensity, but some of them also increase with duration. Sometimes there's a kind of combination of both. And I think like FTP and sweet spot work, as long as you're getting out to, you know, pretty close to like total exhaustion or muscular exhaustion, I usually say an eight to a nine out of 10 RPE is plenty. As long as you are getting out there, you are getting a combination of all these signals, it seems. And so with AMPK especially, you ride harder, you get more AMPK signaling. It's pretty black and white. But other stuff doesn't necessarily follow the same pattern, like calcium redox. You don't get... that much of a stronger calcium signal. Well, you kind of do up to a certain point where you keep contracting and riding and contracting and riding and riding and riding. And initially, there's not much in there. And then there's a little more in there. And okay, signal goes up a little bit, sure. But it's not like you can really like flood your cell with calcium. Like at some point, it's going to be disadvantageous. It's going to screw you. at some point if your cell can't handle the calcium. By the way, it's part of what mitochondria's job is also is acting as a calcium buffer. So we need to be careful about where and how we're riding to progressively overload because if we are going to be riding, you know, riding over under our thresholds is our classic example, right? If we want to improve them. So if like, let's say, We are going to look at like weight training is a good way to do this. Oh no, we're going to steal a term from weight training. Sorry. The stimulus to fatigue ratio. I love this term. And I know you love this term too. Yep. So when you ride over FTP to raise FTP, what's happening? You can ride at like, let's say you're riding at like 102, 103% FTP and you can do like three 10 minute intervals or whatever it is. All right, fine, good. Now you ride five to 10 watts below FTP, how many intervals can you do? Like a lot more. Yeah, like six, eight, and some people could probably do 10 or 12 pretty easy if you give them enough rest or longer even. So the question is, where do you get more adaptation? Well, if we look at, okay, we can do three by 10 and they feel really hard, but we are only getting, you know, two or three, four, five percent more activation. Assuming the power and, you know, signaling correlate like that, I would not assume that, but let's say they do. If we ride, and let's say that's, you know, 105%, you know, 1.05 times three. Now, if we ride at 5% under, and we can do six of these, what's six times 95%? What's six times 0.95? It's like, I don't want to do math. It's 5.7. So you've got 5.7, like, let's say, adaptation units, or, you know, sure, stimulus units, I guess we could say. 5.7 stimulus units versus, like, what's 3 times 1.05? We've got 3.15. Like, we've got almost double the stimulus riding a little less. Yeah, and we've talked about this before, too, just because Volume, like raw number of leg contractions is one of the things that you're after here. So going through and just slamming these like super threshold, but still long-ish intervals is a good way to get tired and it feels like a good workout. So, you know, that's cool, I guess. Yeah, no. And also I think... What all this stuff points to is that we need to think about training status too. And by training status, I mean how much training have you done both recently, like in the last few weeks and months, versus your lifetime as well. So if you are super well trained, all the stuff that you did as a noob, it's no longer going to cut the mustard. You have to ride more deliberately. and you've got to rest more deliberately so your hard days can be appropriately hard. So let's look at recovery rides for instance because I know everybody loves me in recovery rides that I'm always harping on. So if you ride a little bit too hard in order to increase your total kilojoule or TSS or whatever metric you're looking at or just because you're fucking terrified of feeling good then like that Ride is already within your body's capability. And there's nothing progressive, there's nothing overloading about it other than you're tired and it's going to impact all of your other days. And so you're adding fatigue for literally zero benefit. And the same goes for endurance rides. If you're doing two to three hours super hard and the next day you are struggling to do your intervals, like let's say you did... 4x10 FTP, and this next time you want to do 4x12 FTP. The day before you're 4x12, you go out and you smash the three-hour group ride. You're like, all right, 4x12, here I go. And you barely get your third effort done, and you don't even start the fourth, you have gone backwards. Because now you did 36 minutes of time versus 40 minutes of time in your last workout. That's not progressive, and it's not overload. Yeah, I mean, you might, but I feel like when that happens, people trick themselves, oh, well, I got a good workout in yesterday on the group ride. Oh, yeah. Which is maybe, you know, maybe, maybe not true, because it could be hard and still not be getting you what you want or what the goal is. If your goal is increased time at FTP, you want to extend your TTE or you want to bump that FTP up, like doing, you know. five-minute hill smashy smashies for a few hours with your friends is not necessarily going to do that for you. Yeah, or I mean, it may if, you know, kind of early on, or if you are more concerned about your kind of group ride type fitness, then make your group ride your main workout. And if you are on the struggle bus on your group ride because you did too many FTP intervals the day before, all right, now you got to, you know, you got to pull the lever in the other direction. And it's like, just because something feels harder. Like we said with the altitude and AMPK thing, it doesn't necessarily mean it gives you more adaptation. In fact, it's almost a guarantee that if you're putting out less watts for the same amount of time or the same watts for less time, you are not getting the same benefit. Do you remember those altitude masks? Yes, I do. Where I was like, people would wear them like, oh, it makes my workout feel harder. Like, yeah, because you're... Restricting your airflow, so you're making your diaphragm and lungs and things like that work harder, but is that, if you're wearing it while lifting weights or something, your goal there is to lift the weights, it's not to make your diaphragm stronger. Yeah, for sure. And it's also, what was that food-based TSS thing where it gives you extra TSS if you don't fuel your ride right? Oh, yeah, who did that? I forget, but... Fuck that shit. Oh my God. Just, just don't, just don't. Whoever did that, just take it back, please. So the, cause you don't get extra signals like that. You know, you know, if we're gonna really big brain this stuff, you know, we would want signals that we know would potentially augment the PGC-1 alpha response. And so we would be like, okay, maybe riding in the cold. is going to work. Because I saw a paper where it's totally untrained people. Okay, sure, we saw a slight increase in PGC-1 alpha, blah, blah, blah. But, you know, why is that? They don't have much mitochondrial density to begin with on average. So they're not generating heat very well. And so untrained people cannot maintain the temperature homeostasis nearly as well because They're lacking the mitochondria to generate heat. And so that becomes an issue. And now that generates redox stress and cellular stress and big adaptive signal on the other side. But if you are well-trained, you've got plenty of mitochondria. You are going to get a very tiny signal there. And it's also more likely that, practically speaking, if you're riding in the cold, this can cost a lot of people watts. Like in terms of just raw output. And so that's, you know, and like I said before, your muscles know the difference. Or time. So that's not going to help too. Or both. Yeah. And so like adrenaline is a signal here too, right? Because adrenaline increases, noradrenaline, things like that increase as we are exercising and exercising harder. But how do we get ourselves to have more adrenaline? Let's start all of our rides while we're stressed out of our minds. Should we do that? No. Just do a bunch of stimulants right before your ride. Well, other than your heart exploding, I think we've all seen that when we're super, super stressed out, we get like one day of like okay legs and then we are in the toilet until we recover and recovery is very slowed by stress. And so, you know, I wouldn't want to augment signaling this way either. And I've... Train people who have done all this and more, either deliberately or not, you know, people riding in the winter, people training while stressed, and it doesn't elicit better results. None of these hacks or the signal augmentation methods, in my experience anyway, have ever elicited meaningfully measurable improvements beyond what normal training does. And if somebody can do better normal training, like unstressed and in the summertime, They get faster. And this gets to a deeper part of my personal training and coaching philosophy, which is that, you know, just seeing the complexity of the signal chain, the side doors, the back doors, the fact that performance itself is the integration of many, many, many factors, we can pretty easily say that no one thing other than your regular-ass training is going to, quote-unquote, hack the signal chain. You want to hack the signal chain? Make your training better. That's it. There's no shortcuts. It's like burning more fat and skipping carbs doesn't work, doesn't really help. You're going to have a bad time. You're going to have a very bad time. And I Googled, you know, what are people trying to hack this stuff? And some of them are. I saw an article by some bullshit artist with his shirt off, who's obviously on all kinds of trend and roids and shit like that, you know, Russian sports supplements. Low PGC1 Alpha and ways to improve it. And there's like normal stuff in there like exercise, but there's also like 15 supplements and there's genetic tests and things that are going to cost you a lot of money. Fuck that. Save your money. Is money, is it money that he is going to be receiving? I presume he takes a large cut or it's his company or something like that. Like, yeah, like. Weird. Four like genetic tests for PGC1 Alpha, like. Oh, four single nucleotide polymorphisms. Check yourself for these. Like, do any of these actually translate to something we can measure in terms of performance? I don't know. I cannot assume that they do. So I think all this stuff also says we need to be cautious interpreting studies because we saw today that there's not a linear relationship between certain cellular markers. and Improvement of Performance, at least in the short term that we had for these mice. So generally speaking, even in the longer term, studies really need to move past, in my opinion, they need to move past looking for the differences in PGC1-alpha mRNA transcription. Because a lot of the studies, like I said before, I've looked at these and I wanted to include them on the podcast until I saw that this is how they're measuring the effectiveness, in which case they're no longer on the list. We got to get to that point and we got to discuss this. Can we really say anything improved? No. Let's look at some performance. So that's my gold standard. Performance improvements. That's the final arbiter of anything is are you getting faster? And that's the standard that we use at Empirical Cycling. And this is one of the reasons from day one, I called it empirical. Google defines that as I know, right? Just who would have thought. So based on, concerned with, or verifiable by observation or experience, rather than theory or pure logic. You know what it reminds me of is back in the late 1800s, early 1900s, naturalists slash biologists really got into what was described to me in bioclass as ivory tower syndrome. They were like, oh, now we understand the principles of selection and inheritance. We can sit up here and theorize everything and we're going to have a perfect picture of how the world works. If only. Yeah. Biology has come a very long way in the last hundred years. Very, very long way. And so we need to be cautious about getting caught up in ivory tower syndrome ourselves. and it's very easy to do. I've done it many times. That's how I know it's so easy. Or maybe I'm just susceptible to it more than others. I don't know. So this is the basis of all scientific endeavors though, right? Like you make an observation, formulate a hypothesis, test the hypothesis, dig further. Like that's the whole thing that we're all doing. There was this, not to leave, you know, Just to blame the biologist, but there is this famous like We can take it. sort of anecdotal tale about like a former advisor of Max Planck's who in like the late 1800s told him not to go into physics because basically everything had already been discovered by now. That's why there's a Max Planck Institute in Germany right now. Right, exactly, yeah, yeah. And not one named after whoever this dude is. I don't remember his name. But I remember Max Planck's name. I think that's the point, though, isn't it? Yeah, and so for those of you unaware, Max Planck was one of the sort of leading researchers in quantum mechanics in the late 1800s and early 1900s. Professor, whoever, told him this in the late 1800s. It was prior to the discovery of quantum mechanics. And so they were just kind of getting through thermodynamics and all this stuff. And I was like, yeah, we kind of figured out everything except for quantum mechanics and relativity and, you know, those things that he didn't even know existed. So, you know, you can't really fault the guy, but... to be able to say like, oh yeah, we've figured out almost everything. Don't be a physicist. Yeah, except that pesky little thing called light and like, what makes up atoms? What makes up those? What makes up that? How small have they gotten anyway? Nobody's found a string yet, right? No, yeah. Strings are still entirely theoretical. All right, well, they're on my shoes and I think that's what counts. So I think one of the other things that we can take from this is that we can use the lab science for sure to support our training ideas or even to weed out our training ideas and sometimes occasionally even come up with new ones even though I've said on the podcast many times that that in itself is extremely difficult because we start with the observation oh this improves performance let's look at potentially why and then let's look at potentially can we change our training intervention based on what we know about it and you know potentially sure but like it You know, a lot of the time, I hate to say it, it always gets back down to this simple stuff of like rest and progression and overload. Boo, it's boring. Boo. I know, I wish. Buddhist man, boo. I wish to a God I don't believe in that I had something fancier for people on all this stuff. But, you know, at the end of the day, now it's getting to me. So at the end of the day, it doesn't matter how sound the theory is if it does not measurably improve your performance. I think that's the biggest takeaway. And this is how I coach and this is how Empirical Cycling coaches. And it's what lets us be flexible. A combination of experience, which we share with each other as coaches here, and observation with understanding the underlying mechanisms about what will and what might not work. Which we're always still willing to try. It means that we know that there's a lot of ways to achieve the same fitness goals, right? If somebody comes up to us and says, hey, I want to improve my lactate oxidation. We're like, all right, you know, saving the larger discussion on lactate oxidation. There's a billion ways to train that. Cool. We've got a lot of options ahead of us to achieve the same thing. And so we also understand the individual variation. That one person is usually not represented by an average. And so that's why I've been saying to everybody for so long about you as an individual and using your RPE and auto-regulating this stuff and resting when you're tired is a big part of auto-regulation. This is all stuff that the science and the lab stuff doesn't know that. You're stressed at work. You slept like shit last night. You had too much coffee. You've got a job where you work in like a, I don't know, like a level four biohazard place. You're working on anthrax and you can't really snack at like two in the afternoon because you got to like suit down and that takes an hour, like all this other stuff. Yeah. Or like you had like a very manual labor. Job, where, you know, you're actually actively sort of burning matches effectively that you could use later in a workout, but you're doing it to work, you know, accomplish your day-to-day job. Yeah, and I cannot imagine how many tens of thousands of steps I took every single day. It was a lot, for sure. And some days were less, some days were nothing but up and down the stairs, and that sucked. and coaching and all that stuff for you coaches out there and all of you self-coach athletes and I know there's a lot of you out there. It's, you know, it's also not just about power too because when we're looking at performance at events and races, there's more to it than just showing up with an overwhelming amount of firepower. Like if you go to your... Cat 3 race, and you've got a 400-watt FTP, and you can just ride off the front, like, good for you, but, like, you are very quickly going to get to a point where you don't have the skill set to race with that group, and, you know, that's one of the things that we would want to work on, like, hey, go to this hard group ride instead, or, you know, maybe sit in the pack and practice, like, going through it, even though you could ride off the front, like, just hang out with everybody, meet some people, and then you can ride off the front in the last, like, it doesn't matter how far you win by. You can go off the front of the last 2K and they'll be like, oh God, well, he let us think for 50K that we had a shot. This guy again. Yeah, this guy again. Please upgrade him soon. So anyway, like all of this stuff comes down to, you know, did your training make you faster? And you don't need to measure PGC-1 Alpha for that. Yeah. Also, I think within that is like, There are some things that people want to be able to, in research, they want to be able to measure something and there just is nothing, there is no good thing, good proxy to be able to measure to get there, right? Which is why you say ultimately like performance is the ultimate measure. It would be nice, like we said, if you had a blood test for effectiveness of a workout or quality of a workout or, you know. Oh, there's this one blood test we can take to show that you have a higher current level of fitness than you did a year ago, a month ago, whatever. But we don't have that, right? We just have going out and doing the thing. Yeah, and that's, you know, like combination of looking at our subsequent metrics between RPE, heart rate and power, and race results, that's how we do it. That's my standard. That's how I think. Well, it's probably not how everybody thinks it should be done, but that's how we think it should be done. And I also think that that allows us to be more objective in the way that we train people, too. Not to turn this into a sales pitch or anything like that. I mean, I just mean this as like a general training philosophy thing, because if somebody out there is wondering, like, doesn't have a training philosophy yet, just use mine for a little bit until you kind of figure yours out, if you want. Like we can be objective in a good way. And I think a lot of, I think a lot of good coaches do this too, by the way. So we're definitely not just us where, okay, somebody made more power. Okay. Did your one minute power go up? Cool. But did you get to the end of that crit? No? Okay. We got other stuff to work on. It's, it's, it's a very, very simple formula. I know a lot of people who will you know train sprints and stuff and that's one of the reasons I wrote that training too hard for criteriums articles forever ago because I knew people were just smashing themselves to bits I still know people like this and and it doesn't matter how many watts you can do if you are starting from 10th wheel coming out of that last corner in a crit like you could have 2,000 watts you're probably gonna at best get up to like fourth maybe third it's it's not gonna be it's not gonna be a win That's for sure. It's a hard life doing that. It is definitely a hard life. So anyway, do you have any more thoughts or we can get to our couple listener questions because we have some... No, I think it was good. I mean, apologies to people who thought talking about some of the PGC-1 Alpha stuff is a little too in the weeds, but hopefully, you know, again, we're sort of trying to peel back the layers here to see why training and adaptation works the way it works. And so... you know there are some good takeaways here and yet again we kind of find that there is no there is no silver bullet so yeah sadly because otherwise I would sell it and be a rich man indeed all right so let's get so many so many what Ferraris to buy yeah you know I'm a Ferrari guy yeah my collection my butler just got taking care of my collection right downstairs right now Downstairs, just kidding. In the garage hanger unit on the other side of the ranch. So, first listener question. Dynamic between the slow component and local muscular adaptation, like watts versus ventilation. Actually, slow component, usually, if we control for all other factors, it can happen for a couple reasons, but typically the big one is that we are recruiting larger motor units, and they're less well-trained, and they're less efficient. at turning oxygen into power. So they're generating more heat. And to make the same power, they need more oxygen. And that's one of the reasons that the heart rate's going up and the ventilation is going up. So one of the things that happens with this is, well, it could be like hydration, something like that. If you're dehydrating, sure, it's gonna give you problems. But you are getting into larger motor units as you train longer, as you ride longer. And that's one of the things that I like about longer intervals. And so if you are doing an interval of 30 minutes and you are fatiguing by the end, you're probably going to get into some pretty big motor units indeed. And if you keep doing, if you do like six by five minutes and you're not really, you don't really see that much decoupling, then that's a big difference in terms of the motor unit recruitment. So potentially there is a, you know, that could be a proxy for getting into larger motor units if you control for all your other stuff, for sure. 

Let's see. Oh, yes, we definitely touched on this. PGC1 Alpha Expression, Regulation by Temperature, PKA, CREB, and P38MAPK, ATF2, in parentheses, Big Brain. Yes, that is Big Brain. All this other stuff is, yeah, don't bother with it. Just train normally. Let's see. Threshold and sweet spot intervals, are they useful mainly through AMPK and CAMK pathways? No, there are a lot of other pathways, like exercise intensity is directly related to the expression of adrenaline and noradrenaline while you exercise, and it's also related to a larger amount of redox demand and greater cellular stress, so pretty much... Most, or if not all, pathways are active while you're doing threshold and space bite intervals, potentially. Let's see. More watts equals more PGC1 alpha? Yes. More watts equals more PGC1 alpha, asterisk, activation. Through more activation of... Other signals like AMPK and stuff like that responds directly to intensity. The other way to think about it is in terms of motor unit recruitment. I think we've talked on this podcast a lot, where if you are pushing harder watts, you are getting into larger motor units, and that has an effect too. And there's a lot more complicated stuff about, you know, kind of microdynamics of the cell and oxygen and stuff. But generally speaking, that's a good way to think about it. Oh, here's a good one. So here's a big brain. I love this one. Does riding fasted or at least not actively refueling Z2 rides increase PGC-1 alpha signaling? Pretty definitively. Yeah. I wanted to sigh at this, but I don't think I've ever seen a question from this person before. So welcome, Sir Nornick something. Yeah, so definitively no. And actually people have done very direct studies on this about does if you don't eat like after training, does this increase any kind of signaling? The answer definitively in those studies is absolutely not. And so the thing is about fueling, because like we said a million times, it's not about the substrate that you're using. It's about the duration. and intensity of the exercise. And if the exercise is not going to be intense, make it low intensity and go long, big duration. And that's a pretty easy trade-off to make. So yeah, we don't get extra signaling by starving ourselves or any of that stuff. Because otherwise, all those Louise Burke studies on racewalkers and keto stuff, well, I guess that would be carbs and fat, so maybe not those. But yeah, it's pretty definitive that it does not help to rob yourself of food. In fact, it can lead to some pretty bad things, and we've talked about this before, so, you know. Yeah. Okay, next question is, signaling doesn't always equal adaptation. In what setting does it equal adaptation? This is pretty easy. When you get better performance. That's it. When you're faster. When you see that you are faster. Oh, here's a good question. I really like this one. So, can PGC-1 alpha be blunted? Chronic overtraining? Excess anaerobic demand? Not excess anaerobic demand for sure. Usually excess anaerobic demand like sprints and things like that are typically associated with, at least in the moderately and untrained people, a very large signaling response. But it can be blunted because it takes time to repair and it takes time for these things to settle in. And this is why we have rest weeks. This is why we have rest days. Overtraining and excess of, you know, intense sessions and things like that really stop stuff in their tracks. And we went a bit into some of the mechanisms of this kind of stuff in the last Wattstock on redox signaling, which I think was came out last November or something like that. So it's, yeah, the expression can be blunted, I think. if you are definitely training excessively. However, in some instances, more training equals more adaptation. And it's a very, very strange balance that you have to find in your training between watching your performance improve and resting. and that high-intensity, low-intensity balance. And we've talked about this a lot on the podcast up to now, and so we won't have to rehash all of it, but yeah, like if you are not, if you're not getting faster and you're doing all these things, you're like, I should be getting faster, I should be getting more fatigue resistance or something should be happening, and it's not, a lot of the time it is because you're not fueling right or you're not resting enough or you're not progressively overloading your training in a sufficient way. So yeah, it totally is possible. Interesting question. Should endurance cyclists be concerned about the interference effect? Explain where mTOR fits in, please. So yes and no, because the reality of the interference effect, in my experience, it comes... down less to the actual signaling interference, and it comes more down to how the fatigue of certain things affects your other workouts. So for instance, if you do a super heavy lift session Monday morning, then Monday evening, you've got some like threshold intervals, but you are wrecked and you can barely hit your threshold. Oh no. Yeah, this absolutely affects your signaling because it's the same as going to altitude. It's like your muscles are capable of doing it. And I saw somebody put it like this, like if you can do advanced math but you get drunk and you can't do advanced math anymore, did you actually lose the ability to do math or can you just not express your ability to do math? And that's not a bad way to think about it. Yeah, that's an interesting analogy. Was that Coggin? It might have been Coggin. I forget where I saw that. But it's a really good analogy because that's more where I see the interference come from is fatigue, acute fatigue, more than any kind of like long-term, oh, you've been lifting too much here, so therefore blah, blah, blah is whatever. It really comes down to the fatigue and the progressive overloading, for sure. And it can... You know, I've trained some people where we will deliberately use the training interference effect to make sure that somebody's not putting on muscle mass, but it's most of the time, most cyclists are not training in such a way where they're not, they're usually not going to gain much, if any, weight, even if they lift for two, three, four months, like, because it's pretty much going to go away the second you're out of the gym for like three or four weeks, so it's usually not that big of a concern to me. Yeah. I mean, you imagine how long, you look at how long, years and years and years, even with chemical enhancements, it takes some of these pro bodybuilders to add an appreciable amount of muscle mass. And you're worried that like four to eight weeks in the gym and the off season is going to, you know, it's just, yeah. And there's just not enough time. Like you said, the interference effect is real, but there are all these other things that are a much larger effect. Yeah. I think I wrote a Training Peaks article about that a couple years ago now, too. It was something about concurrent training. I have completely forgotten. You remember that one. Wow, I don't. Okay. Now that you mention it, it rings a bell. Yes, yes. If you want to read it, go to theempiricalcycling.com and go under Education and Articles, where I've linked all of our coaches. articles and publications and stuff that you can read, including my own and Fabiano's and I think that's it. I should get Rory's on there unless somebody wants to read his PhD thesis. Okay, last question. Fast start intervals, like starting fast and settling into sweet spot for increasing time at metabolic intensity for adaptation? Not really. I mean, in a way, potentially, yes. Because this is where we have to think about motor units. Because this is basically, to me, if we do a fast start and then settle into sweet spot, what are we going to do next? We're going to start fast again. So now we've made over-unders. And so when I think about over-unders, the unders are Impacted because the blood flow is higher because you are trying to recover your larger motor units that you just used and recruited in your overs. And that can cause some stress on your sweet spot type motor units. And if you do like, let's say three by 20 of over unders or something like that, and you normally would have some Increased Muscle Recruitment for a sweet spot. You're going to have a lot of it for sure when you do over-unders. And so you're getting into larger motor units as you go, and then you're getting into even bigger ones as you do the overs. And so this all kind of has this weird interplay. And the simple way to think about it is just, can I push the overs harder or can I do the entire thing longer? And there may be some benefit there, but at the same time, I usually think about it in terms of do I need to train the larger motor units? Do I need to train this person to push and rest and push and rest? Or like, what are we doing here? Do they just need some kind of fun? Do they hate steady state intervals and they like these better? Then that's fine. But we're not really getting that much better adaptation, it seems like. It seems like in practical terms, it's less about the total Muscle Improving, you know, let's put it this way. If we do 3x20 sweet spot versus 3x20 over-unders, are the over-unders going to get us that much adaptation if we forget about the big motor units? Probably not that much. Does that sound like the right answer to you, Kyle? I think that's the right way to say it. Yeah, I think... Not to say that it is kind of like one of those, you know, big brainy things. Like, what are you trying to get out of it, right? Like, what is the, what is that extra benefit really there, really gonna, you know, do what you think it is doing? Yeah, I mean, do what you want it to do. Yeah, go check out the over-unders are not special. episode of Wattstock and then the subsequent TMT 10-Minute Tips episode after that, which Kyle and I recorded in the same day, which, by the way, probably should not have done. We were loopy by the end of that one. Yeah. But yeah, go check that out. Yeah, like you said, if you like them, and that's great, you know, and if it helps get through the monotony, especially like you're on the trainer or whatever, you just want to, that's fine. That's great. Yeah, nothing wrong with that. Just don't, don't. Kid yourself or lie to yourself that these are extra special. Yeah, for sure. Because I think the person asking the question, he knows it's not about fat and lactate and all that stuff. So he's kind of asking, actually, what is, I think, a good question about do we get extra by doing over-unders in terms of all these other signals? And the answer is it's untested, but probably not. Not to the degree, especially if we do single fiber. stuff, we'll probably see, yeah, probably see a little bit of improvement, but probably nothing that measurable, so if you like doing steady state stuff, and that's fine with you, keep going, if you like over-unders, that's fine, keep going, if you feel like you need over-unders, keep going, if you feel like over-unders are exhausting you, fuck them, forget it, you know. Yeah, or, you know, if you want, if you've got... 3x30 minutes or something like that and then all of a sudden you realize in the second one you're like, man, this sucks. Yeah, add in some over-unders for it and then if that helps you get through it, you know, great. You don't have to, not all of them have to be over-unders, you know, if the goal is just to get this much time in zone and that gets you there, then the average power comes out right, cool. Yeah, I don't even think about it in terms of average power either. I just think about, I honestly, I don't even equate the two. I think of over-unders and like steady-state stuff as kind of separate bins in terms of what to progress and overload. But yeah, I mean, this is just, you know, kind of like my nitpicky way of sorting out training and if something like, something different works for somebody listening. That's totally cool. Keep doing what you're doing because, I mean, this is getting back to the individualization thing. And so now that I am slipping all over my own words, I guess it's time to wrap this thing up. So thanks everybody for listening and we really appreciate you listening. If you like the podcast, please share it. And if you thought it was a bit wordy, well, we're so sorry. Wanted to really, really do PGC-1 Alpha justice. And it's brethren PGC-1 Beta and peace. PCR? No, not polymerase chain reaction. The third one. The lethal one. The lethal one. The lethal one, yeah. That's a good nickname. The lethal one. Like you're an MMA fighter and you're walking out in the... Kyle the Lethal One Helson, here we go. Exactly, yeah. Coming to slay the belt squat. Oh, God. Yeah, so, yeah, so if people want to support the podcast, empiricalcycling.com slash donate, toss us a donation, keeps the lights on, really appreciate it, and if you really want to coach with us, or just consult with us, look at all of your training, we can look at your files, we can answer your questions, we can talk about PGC1 Alpha, we can do all of that kind of stuff, shoot me an email, empiricalcycling at gmail.com, and of course, for students and professional athletes in some extenuating circumstances, we are negotiable in our price, so please reach out, get a conversation started, it is currently mid-February. and a big race season here in the U.S. about to start getting underway. I know down in Australia, it's been underway for quite some time. So if you are looking for a coach or you want to talk to somebody about periodization or anything like that, shoot me an email empiricalcycling at gmail.com. And if you would like to ask a question for the podcast or my weekend AMAs, please follow me on Instagram at empiricalcycling. And with that, we'll see you all next time. Thanks, everyone.