Jay Feldman  0:00  
You've probably heard that cancer cells can't use fatty acids or ketones, so going on a low-carb ketogenic diet protects you against cancer, while transitioning the whole body off to a fuel that the tumor cells can't use, which is fatty acids and ketone bodies. The tumor cells have a bad mitochondria; they can't burn those fuels. They're dependent on glucose and glutamine. Cancer

Dr Seyfried  0:21  
cells can't switch from these fermentation fuels to fatty acids or ketones because their mitochondria are bad. They can't burn fatty acids or ketones. Stephen, you can't believe how people misinterpret information. They see droplets of fatty acids in the cytoplasm. Say, "You see the cancer cell needs all that fatty acid? No, they can't. It's there to protect them. If they try to burn it, they blow this up and die, so there is a storage of fat. They can't burn fatty acids or ketone bodies. So I knew cancer needed glucose, and I knew cancer couldn't burn fatty acids or ketones because this organelle is is broken.

Jay Feldman  0:52  
But is this really the truth? I'll be answering that question in this video, where we'll be going over Thomas Seyfried's claims that cancer cells can only use glucose or glutamine for fuel, and that they can't use fat or ketones for fuel. And we'll also go over whether this is actually supported by the research. Now, this will be a rather technical and research-heavy video because there are some technical claims being made that we're going to be evaluating. But I do think it's worth digging into, and I will make a point to simplify and summarize as we go, and I'll also be doing a shorter version of this video in the future, summarizing the points and keeping it a little bit simpler without all the research. But in this video, we are going to be breaking all of that down. Now, I do want to start by just mentioning a another aspect of the cancer discussion, which Dr. Safery talks about a lot, which is the idea that cancer is a metabolic disease rather than a genetic disease. So in his videos and his you know content, he provides evidence against what's called the somatic mutation theory of cancer, which is the idea that cancer is basically caused by random DNA mutations, and instead he makes the argument that there's metabolic underpinnings to cancer development. He also talks about the Warburg theory of cancer, which is basically that cancer cells have a dysfunctional metabolism and they rely heavily on the fermentation of glucose through glycolysis. That this drives the cancer state, and he also adds in as well those fermentation of glutamine to that picture, and I did want to mention this because this is an area where we do have some common ground. I do agree in general with the metabolic theory of cancer and that cancer is not simply a genetic disease, but we do disagree on some of the details, and that has to do with what's going on metabolically and also what the solution is, because a major part of the solution that Dr. Seyfried recommends is a calorie restricted ketogenic diet. So that's one area where we definitely differ, and most of that comes back to the question of whether cancer cells can actually use fatty acids and ketones as a fuel. So that's what we'll be focusing on in this video. In a follow-up video in part two, I'll be digging into more of the actual diet side and whether a ketogenic diet is actually effective for cancer and all of that. But in this video, we'll focus on whether the cancer cells are actually able to use fatty acids or ketones as a fuel and what is going on in terms of fuel usage in cancer cells and in cancer cell mitochondria. So with that, we have this central claim from Dr. Seyfried, which is that cancer cells cannot use fatty acids or ketones. So as a result, a ketogenic diet, especially a calorie restricted ketogenic diet that provides those fuels, won't feed cancer cells. Tumor cells have all these defects; they can't use the ketones. This is a fuel they can't use because they're mitochondria defective.

Dr Seyfried  3:43  
So I said, why don't you try to grow your cancer cells in fatty acids and ketones in the absence of glucose and glutamine? Well, that's impossible. They don't grow. We showed that yes, cancer cells do take in oxygen, but they're not using it for ATP. You need a good mitochondria to burn ketones and fatty acids. You need a good oxidative phosphorylation system, and the common phenotype of all major cancers is deficiency of oxidative phosphorylation. Cancer cells can't burn fatty acids or ketone bodies. Checkmate.

Jay Feldman  4:11  
So we hear him stating that clearly in those clips, and he's also stated that in his research papers as well. So this is his 2020 paper titled "Consideration of ketogenic metabolic therapy as a complementary or alternative approach for managing breast cancer, and I'll share a quote here that basically breaks this apart in a little bit more detail and gets at what he's what he views as the issues metabolically in cancer cells and the fact that they can't actually use fatty acids or ketones, even though a lot of the research suggests that they do. So, I'll go ahead and read this quote, which states: Cancer cells cannot effectively use ketone bodies or fatty acids for ATP synthesis through oxidative phosphorylation due to defects in the number, structure, and function of their mitochondria. Moreover, ketone bodies and fatty acids cannot be. And thus cannot effectively replace glucose and glutamine as an alternative energy source for cancer. Bartman et al. showed that the major ketone body beta-hydroxybutyrate could not stimulate breast tumor growth in vitro; hence, ketogenic metabolic therapy becomes a putative therapeutic strategy for managing most cancers, including breast cancers. He goes on to state there have been reports, however, suggesting that some cancers, including breast cancer, can oxidize ketone bodies and fatty acids for growth. The uptake of ketone bodies or fatty acids, together with oxygen consumption in tumor cells, is not proof that the ketone bodies or fatty acids can be used to generate energy through oxidative phosphorylation. Fatty acids can stimulate utilization of glucose and glutamine. Also, palmitic acid cannot support in vitro tumor growth in the absence of glucose and glutamine. Hence, it would be helpful to include evidence of normal mitochondria ultrastructure and electron transport chain activities, and reports indicating that ketone bodies and fatty acids are fuels for oxidative phosphorylation-generated ATP synthesis in cancer cells, so a lot there. But essentially, what he's saying is that cancer cells cannot effectively use ketones or fats. They can only use glucose and glutamine through fermentation, meaning not through oxidative phosphorylation. We'll talk about what that means. And there are studies that show that when you give certain cancer cells ketones or fats, it increases growth, and we see increased oxygen consumption, which is normally a sign of increased metabolism. And he's basically saying that this isn't proof that the cells are actually using the ketones or fats for energy, but rather those ketones and fats could be stimulating the glucose and glutamine metabolism in these cells, so it could be increasing energy production from glucose and glutamine. Now you might be wondering why that matters. If fatty acids or ketones are stimulating growth in cancer cells, that's not a good thing. Why does it matter the mechanism through which they're doing that? And then something will break apart a little bit more later, but it has to do with some of the strategies that he suggests, where Dr. Safri basically suggests that with a calorie restricted ketogenic diet, you can lower glucose levels enough to reduce glucose availability for cancer cells. I think that's arguable, but that's one of his arguments. And then pharmacologically, we can prevent glutamine metabolism. And so, if you take out glucose and glutamine from the equation, he's saying that fatty acids and ketones won't actually cause a problem because in all of these studies, it's that the ketones and fatty acids are increasing the utilization of glucose and glutamine, not that the cancer cells are actually using the fats or ketones for fuel. So that's the general argument here, and so we'll be breaking this down, including the studies that he talks about that increase growth in cancer cells from fatty acids and ketones, the studies he cites in favor of his position, his counterargument, and then some other studies that I would say show pretty clearly that his argument is not actually the reality, and that's because we do have numerous studies showing that fatty acids and ketones will not only support growth and metastasis, but they'll actually be used for energy production for producing ATP. So, we'll get into all that, but we're going to do this in order. First, looking at the studies that show that ketones and fatty acids fuel cancer growth and metastasis, then Dr. Seifried's counterarguments, and then the studies that address those counterarguments.

So, before we do that, I do want to start with a general overview of fuel metabolism, looking at how our cells utilize glucose, fatty acids, ketones, and glutamine, because this will become important when we're looking at the studies, because the studies will manipulate these different pathways or talk about certain pathways being active or inactive, and so it's important to have this general framework first, so that we're able to then connect what's going on in the studies with what's going on metabolically. So let's take a look at this diagram. All right, and we can see that here in this diagram showing the fuel utilization of some different fuels that we'll be talking about today, when it comes to cancer cells, so we have glucose, fatty acids, ketone bodies, and glutamine. So starting with glucose here, when glucose enters into the cell, it can undergo glycolysis, which is that conversion from glucose down through pyruvate, which does produce some energy, and this is happening outside of the mitochondria, just in the cytosol of the cell, and then that pyruvate can be converted to lactate. So you can see that there, and this is basically called fermentation. This is fermentation of glucose, where the glucose is getting converted down into lactate. It doesn't involve the mitochondria, and it still leads to the production of some ATP. So this is a process that a lot of cancer cells really rely on. However, in cells with well-functioning mitochondria, pyruvate can enter into the mitochondria, which we can see here, and then it can be converted into acetyl CoA, which then enters into the citric acid cycle, which is linked to the electron transport chain, which is where we produce the. Vast majority of ATP. That's where we produce the vast majority of ATP in the mitochondria, but also just in general in well-functioning cells. That's where we get most of our ATP is at the electron transport chain, specifically at complex five, which is ATP synthase. So that's the basics when it comes to glucose. So then we have fatty acids. Fatty acids are brought into the cell via fatty acid translocase or CD36, which will become important. We will be talking about that because it's relevant in some of the studies. Then, in order for the fatty acid to enter the mitochondria, it has to become fatty acyl-CoA, and it has to be bound with carnitine, which is done by CPT-1. So we can see that there. That's another one that's going to be very important. It comes up a lot in the research, and that then allows the fatty acid or the fatty acyl coa to enter into the mitochondria and undergo what's called beta oxidation. So that's the first steps of utilizing fat as a fuel is beta oxidation, which produces NADH and FADH two, which generally go down to the electron transport chain and lead to ATP production there. Also, in the process of beta oxidation, we get acetyl CoA, which enters into the tricarboxylic acid or citric acid cycle, which produces more NADH and FADH two, which go to the electron transport chain to produce ATP. So we're following that beta oxidation down to acetyl CoA, that into the citric acid cycle, and then we have the NADH and FADH two that are produced, and then that produces the ATP at the electron transport chain. When it comes to ketones, in this case we're starting with beta hydroxybutyrate. It's getting converged to acetoacetate. It's entering into the mitochondria and being converted into acetyl CoA, and then the rest is the same in the citric acid cycle, producing NADH and FADH two, which generate energy at the electron transport chain. And then we have glutamine. Glutamine is especially important when it comes to cancer cells, where that glutamine can enter into the mitochondria, it gets converted into glutamate, and then into alpha ketoglutarate, which is actually one of the intermediates in the citric acid cycle or the tricarboxylic acid cycle, which we can see here, that is showing that it enters in there, and then can run through that cycle and produce NADH and FADH two. There's also a little bit of ATP that gets produced directly here at the tricarboxylic acid cycle, the TCA cycle, and then again, generally those intermediates are producing energy at the electron transport chain.

So this is our general, you know, kind of overview as far as fuel utilization goes in our cells, which will be important because we'll be talking about what goes on in cancer cells and what doesn't go on in cancer cells, and we'll be looking at in some of these studies what sort of experiments they're performing in order to see what things are actually happening? For example, in some studies they'll block CPT one, which will prevent fatty acids from entering into the mitochondria. In other studies, they're measuring certain products of beta oxidation, or they're measuring acetyl CoA, or they're measuring lactate, or they're looking at what's happening at the electron transport chain. One important thing that isn't here is oxygen. So oxygen is used at the electron transport chain, and this is the only place where oxygen is involved. And so one other thing to mention here with this diagram is that Dr. Seyfried refers often to oxidative phosphorylation, and I just wanted to make that part clear, which is that oxidative phosphorylation involves the production of ATP at the electron transport chain. So down at the bottom there, over at complex five, where we see the ADP converting over to ATP, that's one of the integral steps of oxidative phosphorylation. So when he's talking about that not occurring in cancer cells from fatty acids or ketones, he's saying that they're not actually leading to ATP production at the electron transport chain using the NADH and FADH two that are generated from the fatty acids and ketones. So that's an important detail to mention here when it comes to this diagram. So with that, we are going to dig into some of the research that Dr. Seyfried is citing, first starting with the studies that look at what happens when we add ketones or fatty acids to cancer cells, and what happens in terms of tumor growth and metastasis. But first, before we dive deeper into the research, I do want to mention here that as we go through the research, it'll become clear that simply avoiding carbohydrates with a low-carb or ketogenic diet is not the solution. But I do want to mention that that doesn't mean that all carbohydrates are created equal, or that anybody who's eating carbohydrates is automatically on a healthy diet, or that the standard American diet is healthy. I'm not saying any of that. That's because some carbohydrates are incredibly supportive of our metabolic health, while others interfere with proper metabolic function, and that's why I've created the Energy Balance Food Guide, which is a one-page infographic that organizes foods on a spectrum based on how effectively they support your metabolism, and it also has a separate spectrum that adjusts this scale for you in the case that you're dealing with various digestive issues. This is the place I'd recommend you start to optimally support your metabolism. And lose weight, improve your digestion, get amazing sleep, boost your energy, and resolve various other health issues. The food guide makes it extremely easy to get started with a bioenergetic approach to optimizing your health. So click the link in the description or head over to jfeldmanwellness.com/guide to download your free energy balance food guide. All right, so with that, let's dig into some of the studies that Dr. Seyfried was citing, as far as studies that show that ketones and fatty acids fuel tumor growth. And this first one here was citation 162 in that paper. And so this paper is titled "Ketones and Lactate Fuel Tumor Growth and Metastasis: Evidence That Epithelial Cancer Cells Use Oxidative Mitochondrial Metabolism. And they state we show that administration of 3-hydroxybutyrate, a ketone body, increases tumor growth by around fivefold. Both 3-hydroxybutyrate and L-lactate functioned as chemoattractants, stimulating the migration of epithelial cancer cells. Thus, we conclude that ketones and lactate fuel tumor growth and metastasis, providing functional evidence to support the reverse Warburg effect. So, what they're describing here, as far as the reverse Warburg effect, is particularly important. What they're basically saying is that there are cells near the cancer cells that are producing large amounts of lactate, pyruvate, and ketones, which are then being taken out by the cancer cells and being used.

But the important point that they're noting here is that the ketones and lactate, which is less important in you know in terms of what we're focused on right now, but that they fuel tumor growth and metastasis. They also go on to state. Also, we provide evidence for the upregulation of oxidative mitochondrial metabolism and the TCA cycle in human breast cancer cells in vivo via an informatics analysis of the existing raw transcriptional profiles of epithelial breast cancer cells and adjacent stromal cells. So what they basically found was that when they provided ketones or lactate, it increased tumor growth. And in this part, what they're talking about is how they saw increased gene expression for the genes that are specifically associated with mitochondrial metabolism, with oxidative phosphorylation, with the TCA cycle, with mitochondrial metabolism, with the electron transport chain. So that points to increased mitochondrial activity in these cells. So to summarize, what they found in the study is that when they added ketones, it drove tumor growth and it also increased a tendency toward metastasis, but this was adding ketones on top of a like normal nutrient availability. And they didn't actually measure exactly what was happening when they added those ketones that led to the growth, but they did see based on gene expression, it was likely that mitochondrial metabolism was involved. So that's what we're seeing in this study. The next study that we'll go through very briefly is the one that he referenced as 164, which is titled "Targeting Metastasis-Initiating Cells Through the Fatty Acid Receptor (CD36). So this is looking at oral cancer, mouth cancer cells where they initiate metastasis, specifically cells that are more metastasizing and are highly dependent on fatty acid metabolism, and they also mention that this occurs in melanoma and breast cancer. So they state here: palmitic acid or a high-fat diet specifically boosts the metastatic potential of CD36 metastasis-initiating cells in a CD36-dependent manner. The use of neutralizing antibodies to block CD36 causes almost complete inhibition of metastasis. So CD36, we looked at this in that diagram, is fatty acid translocase. So it allows for cells to take up fatty acids. So they found that that aspect, the CD36, was basically crucial for metastasis initiating cancer cells, and when they blocked it, it caused almost complete inhibition of metastasis. So this is showing that the uptake of fatty acids is crucial to metastasis in cancer. They go on to state: we therefore hypothesized that CD36 positive cells might specifically require lipid metabolism to exert their metastatic potential. CD36 positive CD44 bright cells expressed higher levels of three key enzymes involved in fatty acid beta oxidation. Third, depletion of ACSL1, which adds an acyl coenzyme A moiety two fatty acids to activate their oxidation, significantly reduced the lymph node metastatic penetrance of parental OSCC cells and OSCC cells overexpressing CD36, but not primary tumor uptake. So that was a mouthful. But what they found was that there was an increase in beta oxidation enzymes in these metastatic cells. Remember, those are the first steps of using fatty acids in the mitochondria. And when they depleted ACSL1, which is basically required in order for fatty acids to be able to be taken up into the mitochondria, this reduced the metastatic potential of the cells. So basically, what we're seeing here is that beta oxidation, the utilization of the fats in the mitochondria, was crucial to metastasis in these cells. It didn't actually confirm what was happening after that step. What was happening with the acetyl CoA gene? From that beta oxidation, but it did confirm the role of beta oxidation in metastasis in those cells. And then we're going to take a look at the next cited study here, which was 165, a study titled "The Action of Beta-Hydroxybutyrate on the Growth, Metabolism, and Global Histone H3 Acetylation of Spontaneous Mouse Mammary Tumors: Evidence of a Beta-Hoxybutyrate Paradox. And so they state that beta-hydroxybutyrate, which is a ketone administration, accelerated growth of MMTV new NT tumors, and their metabolic profile showed significant increases in ATP, glutamine, serine, and choline-related metabolites. So this is a breast cancer model, and they go on to state some other findings here, but basically to summarize this part, the administration of the ketone beta-hydroxybutyrate increased growth and increased ATP.

They go on to state that cultured tumor cells incubated with 0.5 millimolars of beta-hydroxybutyrate showed beta-hydroxybutyrate uptake that would be the equivalent to 54% of glycolytic ATP phosphorylation and no significant change in glucose consumption or lactate production. So they're basically showing that the cells were taking up the ketone to a level that would have led to the equivalent of a large amount of ATP produced from glycolysis. They didn't actually measure where the ATP was coming from, but they were just calculating what the potential amount of ATP could have been, and that when this happened, there was no increase in glucose consumption or lactate production. So this uptake in this ketone was something that accelerated growth, and we know also increased ATP, as we'll touch on in a second. But we know that this wasn't due to an increase in fermentation of glucose. It wasn't due to an increase in glucose conversion to lactate, and we see that here in Table Two, where we can see that as the level of beta-hydroxybutyrate increased, its uptake was increased, suggesting that the cells are likely using it, and the glucose uptake and lactate production generally decreased. So the fermentation of glucose was generally decreasing as the ketone was increasing, and then we can see that also depicted here in Figure Seven, where they essentially showed that as the level of beta-hydroxybutyrate increased, the amount of lactate produced and glucose consumed decreased. They also found, we can see here in Figure Four, this was in vivo, that there was an increase in ATP levels in the tumors of the mice that were given supplementary beta-hydroxybutyrate. So when they added, like they gave the mice this ketone, it increased ATP levels in the tumors themselves. And so we can see that here in the section that shows ATP, that kind of fourth section here, where that black bar was significantly greater than the gray one, the control one, showing increased ATP in those cells. And they go on to state, taken all in all, it's evident from the cell culture studies that the C new HER2 cancer cells would have substantially increased their ATP output by oxidizing beta-hydroxybutyrate, even when it was present at the low concentration observed in the solid tumors in vivo, and that oxidation of beta-hydroxybutyrate seems to have substituted for oxidative metabolism of glucose. So, what they were saying that basically they found here is that this ketone was taken up by the cancer cells. There was increased ATP seen there. We know it wasn't due to lactate production, but you know they didn't 100% confirm that the ketone was being utilized to produce energy. They're just saying that that's what everything that they found pointed to. That's what that's the direction that all of their findings led to. And we're not going to go through any other studies showing this because there's other research to get to, other things to talk about. But I did just want to note that there are many other studies showing that providing fat or ketones either as a supplement or via dietary change, you know, using a ketogenic diet can drive cancer cell growth and metastasis. And again, these were just a few that were cited by Dr. Seyfried. Now, his counterargument to these is that yes, there were increases in energy production and growth and metastasis seen in these contexts, but that doesn't mean that this was due to converting the fatty acids or ketones into energy, but rather that this was due to other effects of these fatty acids and ketones. Specifically, that they stimulate the usage of glucose and glutamine. So he's saying that the cancer cells aren't actually using the fats or ketones, but when they're taking up these fats and ketones, it's causing them to use greater amounts of glucose and amino acids to produce energy. And so he states that clearly here in that same 2020 paper we were looking at earlier, where he states, indeed, Kuak et al. recently showed that palmitate could increase oxygen consumption rate by stimulating ATP usage and insulin secretion, rather than by increasing beta oxidation. So he's using this as an example here of a situation where cells could see increased metabolism from a fatty acid, even without actually metabolizing that fatty acid into energy. He goes on to state that fatty acids are potent swelling and uncoupling agents that can stimulate insulin secretion and.

Glucose and glutamine consumption, thus making it appear as if tumor cells can metabolize fatty acids for energy. So, we're going to break down a couple of the studies that he cites here briefly, or at least just what they describe. But again, he's basically saying the cancer cells are not actually converting the ketones or fats to energy. Those ketones or fats are just causing increased usage of glucose and glutamine. Again, the reason why this would be important is because if we can get glucose low enough and we can block glutamine metabolism, then providing ketones and fats, hypothetically, if this is the case, wouldn't be driving growth and metastasis. That's why he's making this argument because otherwise, the research is pointing to the fact that these substrates do fuel growth and metastasis. So, looking at the studies here that he cites, at least very briefly, this is the study that he used as an example for the fact that a fatty acid could increase metabolism without actually being used for fuel. It's a study titled "Palmitate is not an effective fuel for pancreatic islets and amplifies insulin secretion independent of calcium release from endoplasmic reticulum. So, in this study, they were looking at pancreatic islet cells, and what they found was that palmitate, which is a fatty acid, increased oxygen consumption and cytochrome c reduction, both of which are signs of metabolism of energy production, but it did so by stimulating the utilization of glucose rather than by actually metabolizing the fat itself. And so they state palmitic acid induced increase in oxygen consumption rate is not due to increased generation of NADH production by the oxidation of the palmitic acid. They later state the most likely factor mediating the increased oxygen consumption rate in response to palmitic acid was ATP usage. So they're talking about a very unique situation here in very specific cells, pancreatic islet cells, and they don't actually sort out what the mechanism is, but they state that the palmitic acid increased the utilization of ATP, which then increased the metabolic rate to make up for that lack of ATP. They don't state how that happened. That wasn't something they explored here. And it's very much worth noting that this is not a study on cancer cells. This is not actually showing this mechanism occurring in cancer cells in response to a fatty acid. He's just using this as a mechanistic rationale for the idea that fatty acids could increase glucose utilization rather than being metabolized directly because it has happened in other cells, in this case pancreatic islet cells. Nothing related to cancer, but just saying that this could be something that happens in cancer cells. So maybe this explains what was seen in those other studies, so this is again mechanistic evidence for the idea that fatty acids could be increasing glucose utilization rather than being metabolized directly. Then he had a couple of other studies, a couple of other citations that had to do with how fatty acids can induce uncoupling, and along with this, they can increase glycolytic activity. The first one here is a paper titled "UCP2 Transports C4 Metabolites Out of Mitochondria Regulating Glucose and Glutamine Oxidation, and so we can see basically a summary here of their findings. Where on the left, part A here, and this is Figure 5A, is basically showing the effect of uncoupling UCP2 on glucose metabolism, where they're showing that when uncoupling is induced, this reduces the oxidation of glucose and increases glycolysis. It increases the fermentation of glucose into lactate, so it's showing that that one side is favored and the utilization of glucose in the mitochondria is disfavored under uncoupling. And then on the right we have 5b, which is showing the effect of uncoupling on glutamine metabolism, which is actually the opposite, where it's showing that under the state of uncoupling, glutamine metabolism is increased, and that involves ATP production at the electron transport chain. Which, by the way, is something that Dr. Seifrieda said doesn't happen, but that's just a mechanism that they're suggesting in this study. So he's using this as evidence to say, in cancer cells that are uncoupled, as a response to fatty acids, which is touched on in the next study, they could just be seeing increased fermentation, increased glycolytic activity, maybe increased glutamine fermentation in the mitochondria as well.

So that's the the first study here, and then the next one is titled Mitochondrial Uncoupling and the Warburg Effect: Molecular Basis for the Reprogramming of Cancer Cell Metabolism. We're not going to get into any details here in the study, but it talks about how fatty acids can induce uncoupling and that this increases glycolytic activity, very similar to that last one. So again, these are studies that he's using as support for the mechanisms that he's saying are plausible, which is that you know the fatty acids are maybe causing uncoupling in the cancer cells, or through some other way they're increasing glycolytic activity. But just because there's increased metabolism, just because there's increased ATP or growth, doesn't mean it's because the cancer cells are actually using the fats as a fuel. So to be. Clear. This is just a mechanistic rationale for that idea. None of these studies show that this is actually happening in cancer cells in response to fatty acids or ketones. The studies didn't show that fatty acid metabolism or ketone metabolism and the ATB production is not happening in cancer cells. He's just saying that this is a possible explanation for why fatty acids and ketones induce ATP production and growth in cancer cells, but again, there's not actually any evidence supporting this outside of mechanistic rationale. But we're not actually seeing any research in cancer cells showing that that's responsible for the growth, metastasis, and ATP production. Now that being said, it is true that in the initial studies he was citing, the ones that show that ketones and fatty acids can fuel growth and metastasis, they were adding ketones and fatty acids on top of a state with other nutrients available, and they didn't actually measure or do any specific labeling or specific experiments to show that the fats and ketones were actually being utilized as fuel to produce ATP in the cancer cells. So that part is legitimate, at least for the research that he was citing. You know, the research that he was pointing to that other people point to to say ketones and fatty acids fuel growth, and so must be used as a fuel on cancer cells. That being said, we do actually have evidence of mitochondrial utilization of fatty acids and ketones in cancer cells, and that they're actually leading to increased ATP production at the electron transport chain. So that's what we're going to dive into next. I'm going to try to keep these studies brief, or at least the review of these studies brief. But there's a lot to go through, and they kind of build on each other. And I do think it's worth exploring the details here because they do make it clear that the cancer cells are actually using these fuels to produce energy. But it requires that we dig into those details in order to understand how they're showing that. So we're going to start out with a couple of studies here that point in that direction and also provide evidence of oxidative phosphorylation in cancer cells, which is something that Dr. Seifrieda said doesn't occur. He said that mitochondria are basically structurally damaged to cancer cells, so they don't undergo oxidative phosphorylation, and that's why these cells have to use fermentation of glucose or glutamine. Now these studies suggest otherwise, and they also suggest that the fatty acids are being used as a fuel. So we'll start with these, and I continue to build our argument from there. This first paper is for 2016. It's a paper titled "Acidosis Drives the Reprogramming of Fatty Acid Metabolism in cancer cells through changes in mitochondrial and histone acetylation. So in this study, they talk about how in acidic cancer cells where there's a low pH, there's a shift away from glycolysis and away from glucose-derived acetyl CoA, which again is is how we utilize glucose in the mitochondria, toward using more fatty acid-derived acetyl CoA for the TCA cycle in the mitochondria, so let's go through some of the findings that they mentioned here. They say we first found that acidic pH adapted tumor cells were more prone to use exogenous palmitate, that's a fatty acid, as a metabolic substrate to fuel mitochondrial respiration, as illustrated by a strong increase in both the oxygen consumption rate and the mitochondrial membrane potential used to reflect the activity of the electron transport chain. So we see that here.

This is Figure three A and 3b, where we can see that palmitate increased the oxygen consumption rate in the acidic cells and increased the mitochondrial membrane potential. Now we already know, as Dr. Seifried has pointed out this doesn't necessarily mean, on in and of itself, that the cells were using the palmitate for energy production. They could have been using some other fuel, and it is important to note as well the increase in membrane potential, mitochondrial membrane potential, points to ATP production in the mitochondria, but doesn't alone confirm that. They go on to state. Finally, to explore the in vivo relevance of targeting fatty acid metabolism, we evaluated the effects of targeting both fatty acid oxidation with atomoxier and fatty acid synthase with the glutaminase inhibitor BPTES on the growth of CH 6.5 and HT 20 965 xenografts in mice. We found that daily administration of 40 milligrams per kilogram of etamoxia rapidly reduced the growth of tumors arising from acidic pH-adapted cells. The combined targeting of fatty acid and glutamine metabolism showed additive inhibitory effects on the acidosis-adapted cell tumor growth. So, this is important here. So they basically used two different substances: one that can block fatty acid uptake into the mitochondria, and the other that can block glutamine metabolism and prevent it from being used to produce energy. And that's the BPTES. Whereas the atomoxir is the one that blocks fatty acid entry into the mitochondria. And so we can see that in this figure here, which basically shows that. The fatty acid metabolism is not just working in terms of tumor growth by stimulating glutamine metabolism, because if that was the case, if we blocked glutamine metabolism and then blocks fatty acid metabolism, there would be no further reduction in tumor volume. But there was. If we look at just BBTES and then you look at the one where they add adamoxia on top. There's a further reduction in tumor volume, so we know that what that means is that the utilization of fat in the mitochondria in these cells, whatever extent was responsible for increased growth or tumor volume, wasn't just happening due to increased glutamine metabolism. And then we know as well they had mentioned in this paper that glycolysis is generally reduced in these acidic cells, so this is also a case where fermentation of glucose or glycolytic activity is unlikely to be increased by the fatty acid metabolism. So it's pointing to the fatty acid metabolism itself driving growth independent of glutamine metabolism or glucose metabolism. And again, what they showed clearly was that fatty acid uptake, fatty acid-derived acetyl CoA, increased oxygen consumption rate, increased mitochondrial membrane potential. All of those things occurred in response to fatty acid exposure, and that it was actually the uptake of the fatty acids into the mitochondria that was at least largely responsible for the growth effects from the fatty acids. Now, all that being said, it didn't confirm that there was mitochondrial phosphorylation going on from the fatty acids, like from the NADH and FADH two that were produced from the fatty acids. But it's definitely pointing in that direction. That brings us to the next study here, which is a paper from 2020 titled glioblastoma utilizes fatty acids and ketone bodies for growth, allowing progression during ketogenic therapy. So this is a very interesting study where they did some in vitro and in vivo experiments. We're just going to be focusing on the in vitro experiments for right now. I'm going to start with this quote where they state palmitic acid supplementation increased U 87 cell growth. These are human glioblastoma cells under 2.5 millimolar or low glucose conditions, whereas ketone supplementation with two five to five millimolar of three hydroxybutyrate promoted cell growth in both the two five millimolar and the one millimolar glucose conditions. And so we can see that here at Figure 1C and 1D and 1C. This is looking at fatty acid supplementation in different glucose concentrations, and the 25 millimolar condition is extraordinarily high glucose.

Not something that's particularly relevant, but in any case, looking at the low and very low glucose concentrations, the 2.5 and one, we can see that as fatty acid supplementation increased up until about 50 micromolars, there was an increase in cell number and an increase in cell growth, and a similar pattern with ketones, where you saw as ketone supplementation increased up to 2.5 millimolars, there tended to be an increase in the cell number in the 2.5 and 1 millimolar glucose concentration. So we're seeing that in these conditions of low or very low glucose, adding fatty acids or adding ketones led to cell growth, which is pretty notable. Normally, that would point to those being used as fuel. Of course, it doesn't guarantee it, but we're going to continue on with some other things they did in this paper. They state to assess a potential role for fatty acid oxidation in gliomas, we treated U 87 glioblastoma cells with the CPT one specific inhibitor etamoxir in vitro and found that it modestly inhibited growth under standard serum free conditions. Importantly, this demonstrates that endogenous fatty acid oxidation contributes to the maintenance of normal in vitro cell growth in these glioblastoma cells, despite U 80 7s having a p 10 mutation with increased p 13k AKT mTOR signaling, which has been previously thought to inhibit fatty acid oxidation in glioblastoma. So this is notable because they used again atomoxir, which blocks fatty acid uptake into the mitochondria. Saw that when they did that, there was a major reduction, and we can see that here in this figure, there was a major reduction in growth compared to without the atomoxir. You can also see that when they directly silenced CPT one, they saw a similar major reduction in growth. Sometimes they do that because etamoxya can have other effects, especially in high concentrations. So this basically rules out that possibility, but basically shows that the beta oxidation of the fats is crucial for the growth of these cells. Now they also did some metabolic tracing studies looking at the fatty acids and what actually happened with them, which are also particularly interesting. So they state metabolic tracing studies indicate fatty acids are oxidized and incorporated into TCA cycle intermediates. So they basically labeled the fatty acids and then looked at what they were being metabolized into, and then they also looked at that in the case. Of controls and then a condition with etamoxia, which blocked the uptake and then metabolism of the fatty acids. And so we'll take a look at what they found here in this figure, where we can see the blue bars are the amount of the labeled fatty acid that's being converted to these products. So the amount of palmitate, which we can see on the top right, was significantly reduced in the group without the edamaxier. So the group that didn't have a blockage of fatty acid metabolism, they had way lower palmitate levels because that palmitate was being utilized or metabolized. And then we can see with all these blue bars, the conversion into acetyl CoA, into citrate, into malate, into succinate, and fumarate was way higher in the group that had functional uptake of fats into the mitochondria, whereas you can see with those black bars, basically all of the fat stayed as palmitate and wasn't converted down in the TCA cycle. So this confirms that in these cancer cells, they're actually metabolizing the fat through the TCA cycle. And then in this paper, they also did some in vivo experiments with glioblastoma in mice, which we'll be talking about in part two, but just as a sneak preview, it showed some pretty negative effects of the ketogenic diet. But so just to summarize what was shown in the study, we saw that fatty acids and ketones stimulated growth of these cancer cells, these glioblastoma cells. We saw that inhibiting the fatty acid uptake into the mitochondria reduced that growth, and then we also saw that the fatty acids were oxidized through beta oxidation and in the TCA cycle in the cancer cell mitochondria. So some pretty strong evidence for the utilization of fats and ketones in these cells, but nothing 100% confirming oxidative phosphorylation at this point, which is basically the ATP production at the electron transport chain from these fuels.

Now there are a number of other tracer studies that have shown TCA metabolism from fatty acids in different cancer cells, including breast cancer, cervical cancer, lung cancer cells, various others. I'm not going to be going through all those in detail, but there are a number of other studies showing this same mitochondrial metabolism of the fats in cancer cells, but again, not necessarily confirming oxidative phosphorylation, which brings us to a couple of studies that do actually confirm oxidative phosphorylation. They don't 100% confirm it with the fatty acids themselves, but they point to it, and then we'll be getting into that with ketones, and then some even more telling studies after that, but we're going to start with this study from 2024 titled "Fatty Acid Oxidation is Critical for the Tumorogenic Potential and Chemo Resistance of Pancreatic Cancer Stem Cells, and they stated treatment with palmitic, oleic, and linolenic fatty acids notably augmented the self-renewal and chemotherapy resistance of CD 133 positive. Conversely, inhibitors of fatty acid uptake, storage, and metabolism reduced cancer stem cell populations, both in vitro and in vivo. Mechanistically, inhibition of fatty acid metabolism suppressed oxidative phosphorylation activity, inducing energy depletion and subsequent cell death in these cancer stem cells; these are pancreatic cancer stem cells. So, quite a lot of conclusions there. Let's dig into some of the details. And one other thing to mention before we look at some of the figures: they also noted increased fat metabolism genes or fatty acid oxidation genes. They also showed increased lipid storage as lipid droplets and indicators of increased fatty acid metabolism. The reason why I just wanted to touch on this is because Dr. Seyfried has mentioned in some of his papers and elsewhere that in the cancer cells they have increased lipid droplets, which is basically a way of storing lipids. And he was suggesting that this is a sign that they're not actually using them; they're not actually metabolizing them. But in this study, they saw increased lipid droplets alongside increased fat metabolism. So it isn't actually suggesting that just because there's lipid droplets, there's reduced fat metabolism. So with that, let's move on to Figure Three in this paper, which essentially links the beta oxidation in these cancer cells with ATP production, but it is worth noting that glucose and glutamine were still available nutrients here, so it doesn't negate the possibility that the ATP is actually coming from the glucose or the glutamine, but it does point to something else that's pretty notable. So there's a couple of things that we see here. This is Figure 3A, where we can see that we're looking at control cells and then cells with ETO. That's etamox here, which is again preventing the fatty acid metabolism. And in this case, they're specifically looking at fatty acid oxidation with this FAO blue experiment, which essentially shows the conversion of fatty acids into acetyl CoA in the mitochondria, and we can see that this was reduced, especially in the CD 133 positive cells with atomoxir, and we can see that there are notable levels of fatty acid oxidation going on, the conversion into acetyl CoA, and we can see that with atomoxier it lowered the amount of fatty acid oxidation. So we're having some confidence. Of acetyl CoA production from fatty acids. Moving on to the next figure here, we're looking at oxygen consumption rate with the control condition and the edamaxir condition. But it's notable that what we're looking at here is not just oxygen consumption rate, but also specifically ATP-linked oxygen consumption rate, and I'll explain in a moment how they did that. But we can see here in those left four columns under ATP linked that there was significantly more ATP linked oxygen consumption or ATP linked metabolism in the control cells, which was significantly reduced with atomoxier. So we saw that when you blocked fatty acid metabolism, you saw less ATP-linked oxygen consumption, which is essentially ATP production at the electron transport chain. That's basically what they saw, and they did this by using a seahorse assay, which you can see here in this figure. Where basically what they do is they block different parts of the electron transport chain from functioning, and you can see where they show the O, which is right before that 20-minute mark, it shows O at the top with that down arrow.

That's when they introduce what's called oligomycin, which blocks ATP production at the electron transport chain. And so, when that leads to a decrease in oxygen consumption, that indicates that this is the amount of oxygen consumption that was actually linked to ATP production at the electron transport chain, whereas the amount that's left after that was oxygen consumption that was occurring independent, basically, of ATP production. And we can see here that in the control group there was a way bigger drop in ATP-linked oxygen consumption than there was with atomoxier, which shows the amount of ATP-linked oxygen consumption that's coming from fatty acids, and that's how they got to those bars that we were looking at earlier, that showed a major reduction, basically a 50% reduction in ATP-linked oxidative phosphorylation from atomoxia from blocking fatty acid metabolism. And then we can see in the next figure, they looked at what happened not by blocking fatty acid metabolism, but by adding a fatty acid, by adding palmitate. And what they found was that when they added palmitate, that's the lightest blue column, there was an increase, and specifically, I mean, in all forms of of oxygen consumption, but this included the ATP-linked oxygen consumption. That's the the first four columns, and we can see that with the palmitate added, this increased the ATP-linked respiration. Now, again, as we've talked about in other circumstances, it is possible in this case that the ATP production is coming from the palmitate or palmitic acid increasing metabolism of glucose or glutamine at the electron transport chain or something like that, but it does confirm oxidative phosphorylation in cancer cells. It does confirm ATP production at the electron transport chain and cancer cells with this sort of experiment. And this is something that Dr. Seyfried has said specifically does not occur. So we're saying that this does occur. We don't have 100% confirmation from this study that is from the fatty acids that the fatty acid-derived NADH and FADH two are producing energy at the electron transport chain. But we do know that is happening, and if that's happening from NADH and FADH two, there wouldn't really be anything in the cells that would cause it to be happening from the NADH and FADH two derived from glucose versus beta oxidation versus glutamine, so there's not really any argument to be made as for why that would happen. The fact that there's beta oxidation happening, that we have acetyl CoA derived from fatty acids, and that we have ATP production at the electron transport chain essentially points to oxidative phosphorylation of the fatty acids, but we'll also look at other studies that essentially confirm it. Before we do that, I do want to look at Figure Six here, which is where they compare etamoxyr with ranolazine, which ranolazine works by blocking one of the beta oxidation enzymes. So this is comparing the effect of fatty acid transport into the mitochondria versus the actual beta oxidation itself, and so what they found here in Figure 6b, if we look at those first two columns, is again the amount of ATP being produced at the electron transport chain, and that this was reduced the same amount with atomoxier as it was with the ranolazine, which points to the fact that it wasn't just the fatty acids being taken up into the mitochondria, but it was actually the beta oxidation itself, the conversion into acetyl CoA and the FADH two and NADH that get produced in that process that was responsible for the reduction in ATP production at the electron transport chain. So again, that does point to mitochondrial phosphorylation of the fatty acids. It doesn't 100% rule out that this was coming from glucose and glutamine. Again, there wouldn't really be any mechanistic rationale for that. So this does point pretty clearly to mitochondrial fatty acid oxidation and oxidative phosphorylation, where basically what we're seeing is. Fatty acid being taken up into the cell, and the beta oxidation both being crucial for ATP production at the electron transport chain in these cancer cells. And that brings us to one more study looking at this with fatty acids.

This is a 2025 paper titled "SLC 25A35 Enhances Fatty Acid Oxidation and Mitochondrial Biogenesis to promote the carcinogenesis and progression of hepatocellular carcinoma by upregulating PGC one alpha, and they state here we found that SLC 25 A 35 markedly reprogrammed mitochondrial metabolism characterized by an increased oxygen consumption rate and ATP production and decreased ROS level via enhancing fatty acid oxidation. Clinically, SLC 25 A 35 was highly expressed in hepatocellular carcinoma and correlated with adverse patients' survival. So SLC 25 A 35 is a mitochondrial carrier protein that's increased in hepatocellular carcinoma, and they're talking about how one of its effects is increasing ATP production and increasing fatty acid metabolism. And they show here in Figure two a couple of interesting experiments that they did. So the first was looking at ATP levels in the context of blocking different parts of metabolism. So they used a pyruvate blocker, which was UK 5099. They used a glutamine metabolism blocker, which was BBTES, and they saw that with both of those, there was no decrease in ATP levels. So you can see that that's the blue and red bars compared to the green bar. So you can see that both the blue and red bars were the same as the green bar, meaning that if glutamine metabolism was blocked, or if pyruvate metabolism was blocked. There was no effect on ATP levels. That generally points to either the fact that fats are the main thing, you know, being used to produce energy, or if you block glutamine, the ability to use pyruvate is compensating, or vice versa. But then they blocked fatty acid uptake in the mitochondria with etamoxier. That's that orange bar, and they saw there was a decrease in ATP levels. Again, doesn't 100% confirm that the ATP was coming from the fat as opposed to the glucose and glutamine, but it does point in that direction. Then they did the seahorse assay, just like in that other paper that we looked at, and they saw the same thing, which was that when they looked at the ATP-linked metabolism. It was reduced to a greater extent without the edamoxir versus when they had the etamoxyr in there, which points to ATP production at the electron transport chain occurring when the fatty acids are being brought into the mitochondria. Then, in Figure C here, they did a particularly interesting experiment where they used hydrogen labeled fatty acids. In this case, it was oleic acid, and they were measuring the amount of labeled water that was produced. And what they found was that when they did a knockdown of the SLC 25 A 35, there was a reduction in the amount of this labeled water that was produced versus when it was intact. Over on the right, we could see an increase in the fatty acid oxidation. So this labeled water is particularly telling because that gets produced when the NADH and FADH two that are produced from fatty acid metabolism go to the electron transport chain and are oxidized, and water is produced there. And if you have these labeled hydrogens on the fatty acid, you get labeled water at the electron transport chain. So the labeled water that they're recovering here is coming specifically from activity at the electron transport chain with the NADH and FADH2 produced from the fatty acid. Now the one thing to note here: this doesn't necessarily mean there is ATP production at the same time, but normally it would point in that direction. If you're having the oxidation of NADH and FADH2 at the electron transport chain, it generally means you're producing ATP there. It doesn't confirm that 100% However, again, looking at the Seahorse assay, we know that there was ATP being produced at the electron transport chain. So in this study, we do have confirmed oxidative phosphorylation in these cancer cells, and we do have you know a pretty strong signal pointing to the fact that this is coming from the fatty acids, and that the NADH and FADH two produced from the fatty acid oxidation are being oxidized at the electron transport chain. Now, one thing that we're not digging into with these papers as much, just because we're hyper focused on this question of whether these cells are using fat to produce ATP or energy and ketones is the fact that they're pointing to this being a pretty crucial process for metastasis and growth in cancer cells.

Not only that cancer cells can utilize fats for fuel, but that it's actually vital for increased growth and metastasis. And as we'll talk about later on, there's actually a lot of studies pointing to the fact that blocking fat metabolism is actually really beneficial in cancer cells and in humans who have cancer. So that's something we'll be talking about later on. But just something that I wanted to mention here before we dig into a couple of studies showing this with ketones. So these are studies pointing to ketone metabolism. In cancer cells, the first one is a 2024 paper titled "Metabolic Alterations and Cellular Responses to Beta-Hydroxybutyrate Treatment in Breast Cancer Cells. They state we showed that under nutrient starvation conditions, treatment with 10 millimolar of beta-hydroxybutyrate significantly increased the viability of MCF7 cells, close to 100% This is very noteworthy. And again, these are breast cancer cells. This is extremely noteworthy because Dr. Seyfried has mentioned that cancer cells can't survive on only fatty acids or ketones. But here, they found that in starvation conditions, so very low availability of any other fuels. I think there might have been very tiny amounts, if any, available. The beta-hydroxybutyrate was able to function as a fuel for the cancer cells to survive. Now that brings us to some other experiments they did that were very similar to the last couple studies on fatty acids, but basically showing the same thing with beta-hydroxybutyrate, showing that it increased ATB production in the mitochondria at the electron transport chain. So we can see that here in Figure 3c, and so I'm going to share a quote describing this, and then we'll take a look at the figure. They state we employed the Seahorse technique to differentiate between ATP produced through glycolysis and ATP produced by the mitochondria. We found mitochondrial ATP to be significantly increased in MCF seven cells in the presence of beta-hydroxybutyrate. We can see that here in Figure 3c, where when the beta-hydroxybutyrate was added, there's an increase in the blue section there, right? The mitochondrial ATP that was produced, and actually a reduction in the ATP coming from glycolysis. So what we're seeing here is not that the addition of the ketone increased ATP coming from glycolysis or fermentation, but rather it increased ATP coming from the mitochondria, generally pointing to it coming directly from the ketone metabolism itself, doesn't 100% confirm it because unfortunately they didn't actually specify for this experiment what the medium was. So we don't know if there was any amount of like glutamine available, for example. We don't know 100% what was responsible for the mitochondrial ATP production here, but we do know that there was an increase in mitochondrial ATP production at the electron transport chain because they did that same seahorse technique, and that happened in this case in response to ketones. And that brings us to this 1981 study titled "Inhibition by Calcium of Oxidative Phosphorylation in Myeloid Tumor Mitochondria. Again, a much older study, and unfortunately, because of that, we don't have a lot of the the details here. But it does confirm the oxidation of the ketone beta hydroxybutyrate, leading to ATP production. They just state this in a quote here, which says these mitochondria have demonstrated tightly coupled respiratory activity by oxidizing either an NAD or FAD linked substrate, responding to regular respiratory jumps after addition of ADP, and showing the expected uncoupling effects of DNP. So what they're talking about here is an experiment where they added ADP and they saw an increase in respiratory activity, which points to ATP production from the different fuels. And they state in the paper that this was tested with beta-hydroxybutyrate, and that they used a medium without any other substrates available. So it's basically directly showing ATP production from the ketone because there was no other fuels available in the medium, and adding the ADP increased respiratory activity. However, they didn't actually show the figure with beta-hydroxybutyrate included here, but basically what they found was that the beta-hydroxybutyrate led to ATP production in the mitochondria of these cancer cells. And there's one other paper showing this as well, which is a 1994 paper titled "Acetoacetate Metabolism in AS30D Hepatoma Cells. And we can see in Table Two here they added acetoacetate, which is a different ketone instead of beta-hydroxybutyrate, and that this increased labeled carbon dioxide production.

And carbon dioxide is produced in the TCA cycle, so this is confirming that there's TCA cycle activity from the acetoacetate, and we can see that here in Table Two, where there's acetoacetate at two millimolars and zero glucose, and we saw the amount of labeled carbon dioxide produced was actually the same as when they had an extra six millimolar of glucose in there. This doesn't confirm ATP production at the electron transport chain, but does show mitochondrial metabolism of acetoacetate. But then in Table Three we see ADP stimulated oxygen consumption, just like we saw in that last study with the beta hydroxybutyrate, and they did it with both acetoacetate and beta hydroxybutyrate individually. They state specifically there was no glucose, no pyruvate, and no glutamine available, although there was an extremely tiny amount of malate. It was 50 times less than the amount of beta hydroxybutyrate and acetoacetate. So really, that wouldn't reasonably be responsible for the amount of respiration, the oxygen consumption that they saw here. And we can see that they looked at regular rat liver cells versus the hepatoma mitochondria. And what they found was that with the ADP-stimulated respiration from beta-hydroxybutyrate, it was close to the amount in the regular rat liver. With the acetoacetate, it was actually three and a half times greater than the amount from the rat liver, suggesting that the cancer cells were actually using acetoacetate much better than regular cells, and that the beta-hydroxybutyrate was used nearly as well. And again, this was looking at ADP-stimulated respiration, which points pretty clearly to ATP production at the electron transport chain. And they did state this in a quote here, where they said acetoacetate-dependent respiration was approximately fourfold greater for AS30D mitochondria than for normal liver mitochondria, demonstrating an enhanced capacity for acetoacetate oxidation by the hepatoma mitochondria. So that was a couple of older studies, basically confirming oxidative phosphorylation of ketones. And then we had that newer study that was pointing in that direction, didn't 100% confirm it. And then here are a few studies. These are the last ones that we're going to go through, but they're really important to touch on because they fully confirm oxidative phosphorylation (ATP production at the electron transport chain from fatty acids in cancer cells) The first one here is a 2016 paper titled "Enhanced Oxphos or Oxidative Phosphorylation: Glutaminolysis and Beta Oxidation Constitute the Metastatic Phenotype for melanoma cells, and so they state here: the 4C11 positive cells also displayed an increased oxidative metabolism, accompanied by enhanced rates of oxygen consumption coupled to ATP synthesis. Furthermore, 4C11+ cells had a higher ATP content and increased succinate oxidation, which points to complex II activity and fatty acid oxidation, and the 4c one one positive cells. These are metastatic cells, so the cells that are most concerning. And they go on to state, in addition to succinate oxidation, the metastatic cells were able to derive extra energy by using palmitoyl carnitine as a substrate. That's basically the palmitic acid fatty acid bound with carnitine, which is what allows it to be taken up in the mitochondria. They go on to state collectively: these results strengthen the idea that metastatic cells distinguish themselves by their unique ability to harness the energy generated by succinate and fatty acid oxidation. By extension, our data indicate that the extra energy originated in the mitochondria endows four C11+ cells with a phenotype that potentially permits them to migrate and invade target tissues. So again, just the larger context here, talking about how the ability to utilize fats as a fuel is actually something that allows for these cells to be effectively metastatic. And they show some of their experiments here, at least the ones we're going to focus on in Figure Three, which looks at oxidative phosphorylation with ATP production at the electron transport chain, both from succinate and from palmitoyl carnitine, and they used a medium with no other substrates. So we can be 100% confident in the succinate-driven ATP production at the electron transport chain and the fatty acid-driven. For reference here, because there's a couple of different types of cells that we'll see in these figures. The 4c cells are pre-malignant cells. The 4c one one minus cells are non-metastatic melanoma, and the 4c one one plus cells are metastatic melanoma.

So again, we're focusing on just the confirmation of this happening, but also there's some interesting takeaways and findings when we consider that broader context, but if we look at Figure 3g here, this is an interesting one. This is looking at the respiratory complex content of the cancer cells. So this is looking at the amount of the electron transport chain complexes that's existing in these cancer cells. This is something that Thomas Seyfried points to as part of the reason why these mitochondria can't use these fuels in the electron transport chain is because of disruptions here or a lack of the machinery to do so. But this is that machinery, and we see not only is there mitochondrial complex availability here, but it's increased in the more metastatic cells. So that brings us to Figure 3h here, which shows the oxygen consumption rate in these different cells, specifically looking at the impact of succinate. So succinate basically increases FADH two, which drops electrons off at complex two. So we're basically looking at the function of complex two at the electron transport chain, and so we can see in the succinate, that second grouping of columns, that of course, when you add the succinate, there's an increase in oxygen consumption. Then, when you add the ATP, there's a far further increase in oxygen consumption, which points to ATP production allowing for continual electron flow. If there was an ATP production, then adding ADP wouldn't allow for any further increase in oxygen consumption at the electron transport chain. And then we have what they show as state four, which is when they add oligomycin to block complex five to block ATP synthase and prevent that ATP production. And when they do that, you can see that major reduction in oxygen. Consumption. So basically, what we're seeing here in this figure is ATP production from succinate in these cells, and in the cells that are metastatic, we see a far greater amount of electron transport chain activity and a far greater amount of ATP being produced there compared to the non-metastatic cells. And then we can look at the same setup in Figure 3j here, except instead of looking at this with suction eight, we're looking at it with palmitoyl carnitine. That's that PC, which again is palmitic acid bound with carnitine, which allows it to enter the mitochondria. So when we have that palmitoyl carnitine there, we can see there's a certain level of oxygen consumption. When we add the ADP, especially in metastatic cells, there's an increase in oxygen consumption, suggesting ATP being produced at the electron transport chain. And then, with the oligomycin introduced in state four, we see that oxygen consumption drop again, confirming the ATP production at the electron transport chain that was happening as a result of the palmitoyl carnitine, and again to be clear, there is no other substrates available in the mediums here in the media. So this confirms ATP production at the electron transport chain coming from fatty acids and coming from succinate. We are going to look at two other studies that also confirm this. Just in case you weren't convinced, this is a study from 2015 titled "AMPK Maintains Energy Homeostasis and Survival in Cancer Cells via Regulating P38 PGC1 Alpha Mediated Mitochondrial Biogenesis. And here we're looking at lung cancer cells. We're going to take a look. We're going to jump right into Figure One, which is looking at ATP levels in these cells from different substrates with no other substrates available in the medium, and so we can see here in Figure One I the relative ATP levels with no substrate available, with glucose available, with glutamine available, with lactate available, and then with palmitate available. Of course, the one that we're most interested in right now is the palmitate, and they're looking at this in different circumstances. They're looking at this in cells that had normal AMPK activity versus ones that had reduced AMPK activity. The AIC that they're adding is increasing AMP kinase, for example, and the OLI is oligomycin. So what we want to zoom in on here is the palmitate part. So if we look here at the palmitate area, that black bar is the control cells. So the control cancer cells, with nothing else being changed, obviously the amount of ATP is way higher with palmitate compared to with no substrate.

We can see that very obviously there is clear ATP production coming from just the palmitate alone, and then in these other conditions, we can see that when AMP kinase activity is increased, there's further ATP production. That's that red bar, which is the control cells along with the AIC that increases AMP kinase, which is something that they talk about, and we'll come back to. They talk about how it increases in the context of low glucose conditions and increases fatty acid metabolism, which we're seeing here increased ATP production from the fatty acids when you have increased AMP kinase. And then when they added the oligomycin, that's that tiny kind of like olive-colored bar. You know, we can compare that to the black bar and see, you know, major reduction in ATP production coming specifically from the electron transport chain. That oligomycin specifically confirms the ATP production coming from complex five of the electron transport chain. When that was blocked, we saw, you know, major reduction in ATP with palmitate available. So this is confirming ATP production from palmitate in these cancer cells, and then I do want to share one quote from this study, which is talking a little bit about the role of AMP kinase here. They state AMP kinase is activated under glucose limiting conditions as well as by ICAR. AMP kinase, by increasing PP 38 and inhibiting mTOR, regulates expression of PGT one alpha, which controls mitochondrial biogenesis in cancer cells and allows oxidative metabolism of non-glucose carbon sources, including glutamine, lactate, and fatty acids, to generate ATP. To restore the ATP pool, AMPK enhances metabolic rate by activating PFK two. So I wanted to share that quote alongside the graph. You know we have clear confirmation of oxidative phosphorylation of the fatty acids here, but also something else which is particularly notable, which is that these cancer cells adapt under low glucose conditions by increasing the metabolism of other fuels. That includes glutamine, it includes lactate, but it also includes fatty acids. And also, it's worth noting the lactate is generally just coming from glucose. So if there's no glucose, the cells will run out of lactate. So, you know, hypothetically, even if glutamine is blocked, the adaptive pathways here are going to increase fatty acid metabolism to generate ATP, so that the cells can continue functioning. That's what this study was pointing to in these lung cancer cells. It's. Part of why you know sneak preview as far as what we'll talk about in the next video, that's part of why lowering glucose I don't think is a is a clear solution here. But in any case, the most important thing that you know as far as staying on topic is we're seeing clear mitochondrial phosphorylation, ATP production at the electron transport chain from fatty acids in these lung cancer cells, and that brings us to the last study we're going to take a look at here, which is from 2023, titled "Mitochondrial Morphology Controls Fatty Acid Utilization by Changing CPT One Sensitivity to Malonyl CoA. So this is looking at hepatocarcinoma and lymphoma cells, and they state fatty acid oxidation was quantified as the etamoxier sensitive component of oxygen consumption rate in respirometry assays. More specifically, edamamexir-sensitive oxygen consumption rate was quantified as the difference between respiration induced by palmitoyl CoA and carnitine, along with ADP, and that induced by palmitoyl CoA-carnitine etamoxier and ADP, whereby the vehicle is void of saturating levels of fuel and consists only of endogenous substrates. This allows for measuring the capacity to only oxidize palmitoyl CoA. Importantly, atomoxy was used at three micromolars, a concentration that blocks CBT one without off-target effects on the adenine nucleotide translocase or respiratory complex one. So I know that was a lot there. They're just detailing what they did here to specifically isolate the ATP production from the fatty acid itself. So they mentioned there was no other fuels that were available. They looked at the amount of ATP production via this assay with and without enamox here, and they looked at the amount of respiration again induced by the ADP, which shows ATP production. So all of this is shown in Figure One.

We're going to take a look here at Figure 1b, where they're looking at the Seahorse assay, and the they made it simple where they showed the highlighted section, which is representative of the amount of ATP that's getting produced from oxidative phosphorylation from the fatty acids. So what we're seeing here, and it's basically the you know very similar in both graphs, but we can see in the top one there's actually a greater amount of ATP production. I'll explain why that is second. But what we're looking at is when they added ADP, and there was no atomox here, we saw a major increase in oxygen consumption rate, which means that the ADP was being converted to ATP. Then, when they added the oligomycin, that was reduced, which shows that when you blocked the ATP production at complex five at ATP synthase, then there was that reduction in oxygen consumption. So that's pointing to clear ATP production from the fatty acid at the electron transport chain in these cancer cells, and again, the fact that this was prevented with atomox here-that difference is what represents the amount coming from the fatty acid. Just to explain a little bit further, the top versus the bottom figure: the top was with mitochondrial fission intact. That process intact, whereas at the bottom, mitochondrial fission was blocked. This is again basically like adaptive responses from the cancer cells that are involved with adaptations to stress, and one of those adaptations tends to involve increased fatty acid metabolism. Something that's shown very clearly in non-cancerous cells, and also shown in cancer cells as well. Okay, that was a lot of research. I know it probably got very technical. So with all that, let's take a look at the original diagram that we had started out with, looking at fuel metabolism in the cells, and we'll summarize what we actually saw in the research. So here's all of the you know different potential fuels, for the most part, that the cell is going to use. So the argument from the beginning from Dr. Seyfried was essentially that the primary pathways for energy production in cancer cells were the conversion of glucose to lactate and the uptake of glutamine into the mitochondria, and then being used just in the TCA cycle, and that the electron transport chain wasn't involved in ATP production. There was no ATP production happening here, and that in cancer cells this was largely what was going on. They're producing ATP from fermenting glucose and lactate and from using glutamine just through the TCA cycle in the mitochondria, he also argued that ketone bodies and fatty acids were not being used to produce HCP generally in the citric acid cycle or the TCA cycle, or again at the electron transport chain. So he was saying that ketone bodies, fatty acids were not actually able to be used as fuels in cancer cells, and instead, what was happening is that when the fatty acids were either introduced into the cytosol or when they were coming into the mitochondria, there was an increase in glucose fermentation or in glutamine metabolism. Same thing with the ketone bond. That basically, when the ketone bodies were entering either into the cell or into the mitochondria, you were having this increase in glucose metabolism or an increase in glutamine production. The studies that we looked at kind of went in order through them, showed a number of things. They confirmed that there was ATP production going on, and that this was dependent on the fatty acids getting into the mitochondria. It didn't necessarily rule out that there was an increase in glucose fermentation or an increase in glutamine substrate level phosphorylation going on. But then we saw other studies that confirmed the involvement of beta oxidation, that that was involved in the growth and ATP production and oxygen consumption, the respiratory activity. Then we also saw studies confirming the production of acetyl CoA from the fats as well. We also saw studies showing this from ketones, or at least it was shown in some of them. I don't know if I actually went through those exact ones.

Then we went through studies showing that the fatty acids were actually being used in the TCA cycle and were actually producing NADH and FADH two, and that those NADH and FADH two products were being used at the electron transport chain to produce water, but they were being oxidized here, and we also saw confirmation that ketone bodies were being used in the citric acid cycle. They were producing carbon dioxide here, and that there was respiration going on in the electron transport chain. We also saw studies that confirmed that there was ATP production going on at the electron transport chain at complex five from fuel in general, we just saw confirmation that this was still that this was in fact going on again, based on that those seahorse assays. Even if there was other fuels available, but that it was being driven by fatty acids and ketones, and then at the end we went through some studies confirming that it was actually the case where we had fatty acids going all the way through, you know, being used in the citric acid cycle, and those equivalents being used at the electron transport chain, actually leading to ATP production here, and the same with ketone bodies. So we had full confirmation of the mitochondrial phosphorylation of both fatty acids and ketone bodies in cancer cells. So to conclude here, I do agree with Dr. Seyfried that cancer cells do generally have impaired mitochondrial function, and I do agree that this is not simply a genetic disease, and that there are very, very important mitochondrial and metabolic underpinnings. But based on the research, this does still involve fatty acid metabolism and ketone metabolism and mitochondrial phosphorylation of both of these to produce ATP at the electron transport chain in the mitochondria. In addition, something else that was being shown throughout these studies is that many cancer cells are highly adaptable to using different fuels. This is something that's really important to consider when we're looking at how to actually address cancer, and something we'll be talking about more in part two, and the last really important piece here is saying that cancer cells can't use fatty acids or ketones as a fuel in the mitochondria to produce ATP is not true, right? That's not actually what the research tells us. The research tells us very clearly that cancer cells do, in fact, do this. All right, we're going to end this video here, this very dense video, and pick back up in part two, which will be much less research-heavy, much less technical, and we'll be talking about the implications here. So we'll be talking about whether this means that a ketogenic diet is never helpful for cancer. We'll be talking about whether a ketogenic diet effectively starves cancer of glucose. We'll be talking about what happens to cancer if you block fat metabolism. What happens to cancer if you improve glucose metabolism? And we'll be talking about what all of this means in terms of the optimal diet for cancer, including how I approach this with my clients who have cancer. And as we've discussed throughout this video, it is clear that it's not as simple as carbs are the bad guy feeding cancer, or fats are the good guy or the bad guy feeding cancer either. Both are important fuels for us, and both can be used by cancer cells in certain contexts. And avoiding either of them is not the solution for fixing the metabolic state underlying cancer. But that doesn't mean that it doesn't matter what we eat, or that all fats or carbs are the same in terms of their metabolic effects. Some are incredibly supportive of our metabolism, while others interfere with proper metabolic function, and that's why I created the Energy Balance Food Guide, which I mentioned earlier is a one-page infographic that organizes foods on a spectrum based on how effectively they support your metabolism, and it also has a separate spectrum that adjusts this scale for you in the case that you're dealing with various digestive issues. The food guide makes it extremely easy to get started with a bioenergetic approach to optimizing your health, so click the link in the description or head over to jfoldmanwellness.com/guide to download your free energy balance food guide.

And eating the right foods, which I outline in that guide, can make a big difference, but it may not be enough for reversing metabolic issues. So if you're dealing with any metabolic issues, whether that's insulin resistance, auto. Immune conditions, insomnia, trouble losing weight, or even cancer. Head over to jfeldmanwellness.com/call or click the link in the description where you can sign up for a free call with a member of the Jay Feldman Wellness team who can provide you with insights on your individual health issues and make sure that you get the support that you need on your health journey, whether that's one-on-one coaching, group coaching, or our energy balance course, we've helped people reverse everything from insomnia to fatigue and brain fog to severe digestive problems and everything in between, and we'd love to help you do the same. So again, head over to jfeldmanwellness.com/call, and we'd be happy to help you out. All right, that's everything for this video. Let me know what you think in the comments, as always, and I'll see you on the next one.