 Thank you. There are a lot of good reasons for thinking that ketogenic diets are going to be valuable for treating and preventing cancer. Unfortunately, we have very little data that will support those ideas. And so what I'm going to present to you is kind of energy metabolism 101, which outlines what we're thinking about and where we think we could attack cancer. And I'll present a little bit of the approach in my lab. Unfortunately, in the end, I think that ketogenic diets for cancer is somewhat like what somebody said about military intelligence. It's more what we aspire to than what we have. So the work I describe is the principal investigator is Dr. Eugene Fine, who's at Einstein. And I'm at Downstate Medical Center associated with nutrition and metabolism. I left off the penzoil sticker. But the real sponsors for this are some of the people in the audience. And because the work I'm describing was funded directly or indirectly by crowdsourcing on experiment.com. So we're very grateful to a lot of people who donated and who brought to the attention a donor who wants to remain anonymous but is supporting our work. So this is Gene's work. And in looking at ketogenic diets specifically, focusing a lot of cancer research is in the direction of metabolism rather than focusing on genomic or control of expression. And so we're really going to ask what's wrong with metabolism in cancer cells, which involves, again, outlining what is normal metabolism. And the Warburg effect is a major principle. And I'm going to tell you some of the terms that are used in particular. Aerobic glycolysis seems like a contradiction in terms since glycolysis is always anaerobic that it doesn't use oxygen. So what the term means is simply that cancer cells tend to use, run glycolysis even if they have enough oxygen not to. So that's what the term is. So basically I'm going to maybe call this Warburg 101. Now the general sound bite on the Warburg effect is that cancer cells use glycolysis in place of anaerobic, in place of aerobic metabolism. That's not quite right. Warburg himself recognized early on if you want to kill cancer, it's not enough to deprive it of glucose. You have to knock out oxygen as well. So let me give you some background first and then I'll run through metabolism as we see it. So this is Gene Fine's 2012 study and he had 10 advanced cancer patients. You had to have failed or refused chemotherapy to get into the study and it was a safety and feasibility study and the patients did okay. In addition, several of the patients had showed stable disease or a partial remission and those are the people who had the highest ketone bodies and you can see the blue had higher levels of ketone and this was not due to differences in either how much calories they took in or how much weight they gained or lost. The goal was of course in a cancer study to encourage the patients to stay at a constant weight but they did actually lose weight which has always been to me one of the arguments for a low carb diet is common when you try to maintain weight. It's hard to do that. And what was found is that the insulin and ketone bodies moved in the opposite direction so the patients with the highest ketone bodies had the lowest insulin. Now we don't want to make too much of this. This is a very small study and there's only 10 patients and 30 days but we do want to look at the cellular effects and we took a number of different cancer lines grown in culture and treated them with acetoacetate. Acetoacetate is, I put down at the bottom some terms that either didn't define or may not be familiar with. We had two breast cancer lines and five colon cancer lines and we treated them with acetoacetate which is one of the ketone bodies. The more common beta hydroxybutyrate was not as effective as what I'm going to show you and this has been observed in other places and the defect may be in the interconversion of the two ketone bodies. In any case, what we found is that both ATP, the levels of ATP in these cells in the cancer lines was reduced and this correlated closely with the amount of cell growth so the cells were inhibiting growth in lower ATP. The controls were normal fibroblasts and those are on the left side of the dotted line and you can see that the red and blue bars correlate well and even in the published paper we somehow neglected to say which is which but maybe that makes our point. In any case, the levels of protein were constant for the different effects of ATP so this is not a general, we weren't killing the cells, this is not a general disturbance. The cancer cells overexpressed uncoupling protein too. Now, this is, to be precise, this is a marker that's common to cancer cells. The background is that uncoupling means separating active metabolism from actual ATP generation. In other words, you're running metabolism but you're not getting any usable energy out of it and uncoupling protein one is the naturally, a naturally occurring uncoupling protein and it, what I mean is it uncoupled cells. Uncoupling protein two has a very close homology with uncoupling protein one and we're not sure whether or to what extent it's a real uncoupler. It may do something else, it may be a transport protein so the bottom line on that is just when you thought it was safe to go and look at uncoupling but the uncoupling protein two and ATP are correlated inversely and so it's not excluded that there's a direct effect in cutting off the expression of energy in a usable form as ATP. It is well known of course that cancers are glucose avid and that's a hallmark. Related to that people often say that you can starve the cancer for glucose by not administering carbohydrate but that of course does not work partly because blood glucose is regulated, dietary glucose will not necessarily lower and in addition the cancer cells overexpress what's called glute one which is a protein that picks up glucose with high affinity and there is a particular marker for this, the agent called cytocholazone B does knock out the, I'm going to see if I, do I have a pointer here? Well you can see that, oh okay, this. So you can see that cytocholazone B will knock out half of the glucose, cytocholazone is in the black and in the cancer cells the treatment with acetoacetate is almost as good but it does not have the same effect in the normal fibroblasts. So the question is what goes on in energy metabolism, what is the function of the ketone bodies in controlling it and finally we want to ask how can we get at this experimentally, what I'm going to show you is an outline of metabolism and we're going to ask how could we break into this outline and figure out which parts are really relevant. Now ATP you know is considered a high energy compound, it's really that it takes place in a high energy reaction that is, it can be broken down and is used to run and metabolic reactions that require energy. So I'm going to take the black box approach. Now in the black box approach or systems approach what we do is we, if we don't know what the underlying mechanism is we look at the inputs and look at the outputs and try to deduce what's going on. It's a approach that's favored by engineers who are the people who are most unhappy if they think they don't know anything at all. So we pretty much know what the black box of life is. You put it in food, I'm going to emphasize for a start glucose, you put it in oxygen, you get CO2 in water and somehow this allows you to generate the high energy ATP. So what you usually do is we can deduce here that we're looking at oxidation reduction reactions which would bring you back to general chemistry. The specific thing that I think is important that I won't emphasize too much is that oxidative metabolism can generate unwanted or possibly wanted very reactive oxygen species. And those are usually just indicated simply as ROS or reactive oxygen species because they're a little hard to define in what their mechanism is. So let's look inside the box. The first thing we look at is the process known as glycolysis. The name tells you what it does. Lysis means breaking and it breaks down glucose and somehow that gives us a certain amount of ATP. This is an important note that acid names for carboxylic acids and the salt name are used interchangeably. So glycolysis breaks glucose down. Glucose is a six carbon compound. It usually folds up into a hexagonal structure. And the effect of glycolysis is to generate two, three carbon compounds. So it splits glucose into these two, three carbon compounds. The name pyrovate also may tell you something. Pyro, of course, is the same root as fire as in pyromaniac. And uvo in romance languages refers to grapes. The uvula is the grape shaped thing in the back of your throat. So this is, glycolysis is about firing grapes, in other words, fermentation. So this is the process by which fermenting bacteria carry out metabolism. Alcoholic bacteria will convert pyrovate to ethanol. And the bacteria that make your yogurt will convert it to lactic acid. And we'll see, you probably know that humans can do that, too. So this is the major fate of pyruvic acid in cells is that they are further oxidized to carbon dioxide and they can be converted to carbon dioxide and acetyl-CoA. Acetyl-CoA is a derivative of acetic acid which you may remember from freshman chemistry and is the substrate for the TCA cycle. TCA stands for tricarboxylic acid cycle or also called citric acid cycle or the Krebs cycle. Krebs called it the TCA cycle and that's what we'll use. The effect of the TCA cycle, if you've studied it, you remember the horrendous collection of transformations that you had to learn. But the net effect is that the Krebs cycle converts oxidized acetyl-CoA to CO2 and all those things in the TCA cycle are really carriers more than intermediates. In any case, the oxidizing agent is a compound called NAD and its product is called NADH and it goes into the third of the inner black boxes and where it gets reoxidized back to NAD. This is the oxidation in the electron transport chain is controlled by molecular oxygen so this is where oxygen actually enters into the black box of life and this is where you get most of the ATP. You can get almost 10 times as much as you can get from glycolysis. This is the key slide in that it separates the way in which energy is obtained aerobically that is with oxygen or anaerobically with pyruvate which under those conditions is converted to lactic acid or lactate. This is the starting point and what the Warburg effect is really about is somehow crossing that line didn't go right. We're getting too much lactic acid not enough carbon dioxide. But it's worth looking at this for a minute and recognizing that we still have the same black box of life. You put in glucose and you put in oxygen, you get out CO2 in water. They're separated and the oxygen never sees the food so it's important to recognize that. Now what Warburg measured was lactate and CO2 and he did tissue culture, had cancer tissues and showed that they generated a far greater level of lactate compared to CO2 than normal tissues. This was pursued by the quarries. You may be familiar with the quarry cycle which explains how the liver and muscles can cooperate to run anaerobic glycolysis for its speed in rapid exercising. Girdy quarry was the first American woman to win the Nobel Prize although she was born in Czechoslovakia. This is the commemorative stamp. It actually had a misprint. The structure was not right. This is a corrected version. If you have the original stamp you should hold on to it and it could be worth thousands like the Mozambique purple. What she did is she had a chicken with a cancer in the forelimb and she intubated that she stuck a tube into the cancer forelimb and also into the one that was normal and she compared the blood from those two points in the chicken. What she found is that when she measured the lactic acid in the two veins, lactic acid was always favored in the tumor vein whereas CO2 was always favored in the normal vein and this constitutes a demonstration in vivo of the barberg effect. This is again our working vision of the black boxes. One more thing though is there is another component. One of the components of the electron transport chain is the particle known as the ATP switch. The name says that's the particle that actually makes ATP. It's driven by a high energy state that involves the membrane of the mitochondrion. I didn't say this but you probably know that dotted line was really separating the glycolysis in what's called the cytosol of the cell from the mitochondrial effects. The ATP synthase is the place where the high energy state is actually transport of hydrogen ions across the membrane. That high energy state is dissipated and returns to normal and the energy in returning the membrane to a stable state is what drives ATP synthesis. Here's a biological version of the black box. What it emphasized of course is oxygen coming in but rather than lactate going out H plus lactate is, lactic acid is an acid and so you can measure the acid effect. The original effect of the barberg effect was to think that the mitochondria is damaged but that's generally not true. Something's wrong and the question is where is the problem? Well it could be in the uptake of glucose. We know that we can inhibit that with ketone bodies. It could be in pyruvate and I'll emphasize in another slide that that's the big step from pyruvate to acetyl-CoA. That's where metabolism is regulated substantially and it could be in electron transport. These are just obvious questions. Basically we want to know what's going on. I've separated black boxes into two tissues and in the liver acetyl-CoA is the substrate for the TCA cycle but it's also where the ketone bodies are made and transported. The net effect is to understand what ketone bodies do is they carry acetyl-CoA to the tissues. If we think that we have two kinds of energy substrates. We have glucose and we have acetyl-CoA. The liver transports the acetyl-CoA in the form of this dimer, either beta hydroxybutyrate or acetate and then the tissues will carry this back to acetic acid and the energy will be used for the TCA cycle. This is consistent with the general view that the liver is the kind of command center for metabolism and the tissues are consumers. You can't go back to pyruvate and if you want glucose from pyruvate you have to put in something else which is proteins. But let's focus just on the acetyl-CoA and pyruvate dehydrogenase is the enzyme that converts pyruvate to acetyl-CoA and it is the big control center. It is the big traffic cop. And the main thing is that it has extensive feedback inhibition. So just as maybe a digression it is simply that if you have a lot of energy coming from fat, for example if you are on a ketogenic diet, PDH will see that you don't waste any pyruvate in running energy and shuts down the conversion. Pyruvate of course will be used for gluconeogenesis. So the crowd sourcing allowed us to buy this instrument. This is Anna Miller who does most of the work in the lab and what she is holding is a micro carrier sort of tissue culture sampler that is very small and basically what that is is a Warburg meter. And so the device will carry out Warburg experiments with high throughput. Okay, so where is the problem here? How are we going to find this out? And what you do in the, it is called a seahorse analyzer. Nobody in the company could tell me why it is called seahorse, but that is what it is called. And what it does is it can administer inhibitors. So one way at getting at a complex system like this is to inhibit each of the steps and see what happens. So I am going to show you the inhibitors. We are drifting into a sort of complicated metabolism but bear with me. So one of the simplest inhibitors is a ATP synthase inhibitor called oligomycin. The ATP synthase is called F1FO. FO stands for oligo. The F1 is simply historical. It is the first thing, the first fraction that came off the mitochondrion when they sonicated it. So oligomycin is very useful. Because it will shut down the whole business. If you can't make ATP, you are not going to generate a high energy state. If you can't make a high energy state, you are not going to run the Krebs cycles. Everything is going to stop. We can also put in a variant of glucose, two deoxyglucose, and that will stop glycolysis cold because there is no substrate. That is obvious enough. And I won't emphasize this, but there are a whole bunch of different inhibitors that will target the members of the electron transport chain. If you are a gardener, you probably know rote known as an insecticide. The important one, though, is the uncoupler. And the one that is used is FCCP. And what it does is it breaks the high energy state. So what happens is that it is now, whatever you do, whatever energy you generate, you are not going to be able to use it to make ATP. So there is no ATP. But electron transport can continue to run. In fact, it runs better because it is not locked into further metabolism. So the seahorse goes through a bunch of these and we try to make a deduction from that. To explain the inhibitors to medical students, I usually use this slide, the car analogy. And in the car analogy, the engine takes in oxygen and takes in fuel and generates energy in terms of the movement of the driveshaft, of the crankshaft which is coupled through the clutch to the drivetrain which then turns the wheels, which by analogy is the synthesis of ATP. So this is how you use the engine to move the car. Now, if you put a block under the wheels, that is like putting oligomycin into the mitochondrion. The wheels stop, the drivetrain stops, the engine stops because it is stalled. It cannot run. So everything shuts down with oligomycin. On the other hand, if you put in an uncoupler like FCCP, that is like putting the car in neutral because what happens now is you can race the engine but you are not going any place because the clutch plate is separated. I point out that in German and probably several other languages, Kuplung is the word for clutch and so the clutch is the coupling. So that is what we are going to do now is let the seahorse run these inhibitors and see what it looks like. Okay, this is the slide for the next hour of the talk which I think has to be next year. I will just briefly tell you what happens. What you are looking at there is lactate and oxygen uptake. The open circles are the cancer. It is distinctly different in the seahorse from normal cells. The filled cells are when we add acetoacetate in advance and I will just get you the last summary slide. The bottom line is what this is showing is that acetoacetate is inhibiting both the oxidative part and the glycolytic part and the ketone-treated part is substantially uncoupled and the cancer cell is similarly relatively uncoupled and I'm going to have to ask you to wait for next year for the rest. That's it, thank you. Thank you doctor. Questions at the microphone please. Hi, what is your opinion to use a cytogenic diet and hyperbaric oxygen to treat cancer? We have limited data but of course Dom D'Agostino has done very impressive work showing that the combination of hyperbaric oxygen and ketogenic diet will be valuable. We only have first principle. I don't think we know what will happen. I'm afraid that my opinion on a lot of these ideas which are very good is if you look at the big picture the glass is more than half full but we don't know the answer. There is the problem of course that oxygen will increase the oxidative component of the metabolism and what I didn't show you is that part of the running the electron transport chain involves the generation of the reactive oxygen species. Now what is that doing? Well we don't know what it's doing because these are very highly reactive compounds. They may be deleterious to the cell but on the other hand they may be scavenging uncoupling protein too. We don't know that uncoupling protein too is harmful. We don't know that reactive oxygen species aren't a component whereby cancer cells keeps itself alive by not letting uncoupling protein too get out of hand. I know that's an unsatisfying answer but we have to recognize that both of those are reactive agents that we don't know about. Thank you. Thank you that was terrific. My question is after this discovery of the DNA obviously the Warburg effect kind of took a back seat in a lot of cancer studies and it was Pete Peterson who kind of picked up I think in the 70s and did some studies with the mitochondria and how the impact of cancer and the function of the mitochondria and concluded that there was a defect within the mitochondria but you just said that there really was no correlation between the functioning of the mitochondria and cancer. I'm just curious as to how Pete Peterson's work dovetails with your own work. What I said is that there was the notion that they were damaged possibly physically but the mitochondria are frequently intact. There are cancers where clearly there's something wrong with the mitochondria but functionally there's clearly something wrong. These are not normal functioning mitochondria as far as we can see. Okay. But it's not like the mitochondria and fell apart. Right. Okay, thanks. Hello. On your slide that's up right now in section two are you showing that the combination of the uncoupler and the ketones are more powerful than one or the other? What happens if you just add ketones and don't have that uncoupler? In this experiment I don't want to generalize because we're this kind of... Well you can see there's some variation you have to do. You can see what this is is the basal uptake of oxygen. It's much greater in the cancer cell than the cancer cell that's been treated with acetoacetate. Similarly there's a reduction in the lactate part that is the glycolytic part. So the inhibition that we saw in the gross sense has two components. As to which is... Well to me the major piece that's surprising here is when you... What a cancer cell does and this is one variation on the Warburg effect is at this point we put in oligo. So that's the block under the wheels of glycolysis. The cancer cell compensates tremendously in running glycolysis now to make up ATP. So if we had an ATP curve here you would see that it didn't change at all. But as far as we can see acetoacetate is knocking it out. But this is not the take home message. What I'm showing you is the method. You have to do a lot of experiments from various angles to be sure of that. St. George said today we celebrate tomorrow we do the controls.