 First of all, thank you for the opportunity to present here today. Actually, I found out about the opportunity that I can present a couple of days ago. So I decided that I will talk about mostly today my journey about ketone research during the last 12 years, highlighting the main research findings and the lessons that I learned over these 12 years. First, I need to mention that the information here is not a medical advice. And I also have to mention that I'm an inventor of several patents related to this research, and those patents are owned by the University of South Florida. And I have to disclose I'm a co-owner of ketone technologies and owner of Odysseus Nutrition. So my story started 12 years ago when I moved to the United States after I was hired by the CEO of the Bird Alzheimer's Institute at the University of South Florida. So I arrived with two suitcases with a neuroscience PhD and hope that I can make a big impact, a good impact. The first time when I was exposed to ketones or ketone research was through a hyperbaric experiment when I met my now husband, Dominic Dagostino. At that time, his research was mainly focusing on scuba diving related problems and we connected through scuba diving and as soon after we started working together. At that time, his research was mainly focusing on central nervous system oxygen toxicity which we could simulate in hyperbaric chambers using laboratory rodents where we could increase the pressure, increase the oxygen concentration and simulate these CNX oxygen toxicity seizures without actually putting the rats underwater. And the goal here was to predict and prevent these seizures to restore the brain energy metabolism. So when do people get CNX oxygen toxicity seizures? For example, when they are special operation divers using rebreaters also when people are getting hyperbaric oxygen therapy for example for wound healing, for diabetic wounds and also people are doing space simulation missions underwater and they need to do decompressions. So if you think that these scenarios are very extreme and you want this information just bear with me. The goal here was to preserve the brain energy metabolism. So prior to 1967, it was thought that the brain can only utilize glucose as a fuel. So these fine people in Harvard did a study by starving people and back then it was possible. And they found that after few days of starvation the brain actually started to use a ketone body called beta hydroxybutyrate or BHP primarily and not only glucose as a fuel. They not only starve these subjects but they also administered insulin quite high dose of insulin and they induced severe hypoglycemia. And interestingly they found that the ketones preserved the brain energy metabolism even during severe hypoglycemia. Indeed the ketogenic diet is also used for epilepsy. In some cases sometimes with drug refractory epilepsy it can be very helpful and especially for pediatric patients. You can find more information about this at the Charlie Foundation website. And just a little overview. I know that many of you are familiar with ketones but maybe some of the viewers are not. So just a short overview. How can you get into ketosis? You can either do fasting or starvation, calorie restriction or use the ketogenic diet which is a low carb, high fat diet. So your body will produce ketones. But for some people this is difficult to sustain. They don't want to starve. They don't want to follow a restrictive diet. So for those of us there is another option. Exogenous ketone supplements and precursors. For example ketogenic fats such as medium chain triglycerides, ketone salts which are beta hydroxybutyrate BHP molecules bind to electrolytes and ketone esters. So these ketone molecules, we know three of them and beta hydroxybutyrate is the most abundant. These molecules can then fuel different tissues in the body including the muscle, the heart and the brain. And in the brain it's not only fuel but it's also served as a signaling molecule. In patients with Alzheimer's disease people have decreased glucose utilization in the brain. And in some cases the ketones can restore the brain energy metabolism. So going back to these hyperbaric experiments, the first experiment we used two different kinds of ketone supplements. Butane dial and one tributane dial, acetyl acetate diester or I would just call ketone ester. And indeed the ketone ester was very effective to delay the latency to seizures which was very promising. So later I repeated some of these experiments on more age-appropriate, older reds that age resembled more like a middle-age human. And I also tried different combinations of the ketone supplements. For example ketone ester mixed with MCT or ketone salt mixed with MCT or ketone ester in half dose. So interestingly I found that in this age group of the rodents the ketone ester MCT combination was the most beneficial. And not only the latency to seizure increased but also the seizure severity decreased in these animals. The other interesting thing that we found is that while in the first experiment the beneficial effects were only correlating with the acetyl acetate levels. In these experiments the correlations with the beneficial effects were with the acetyl acetate and beta-hydroxybutyrate as well. So how can ketones restore the brain energy metabolism? We now know that ketones improve mitochondria function. They increase ATP production in the mitochondria when we compare it to glucose. And not only that, they also lower ROS production. And ketones can also serve as signaling molecules influencing the expression of different genes. So based on this idea that ketones can improve energy metabolism. I wanted to study how does it translate in animal research, how can we see the benefits actually. So I started doing motor function research on the laboratory rodents. I used Accelerating rod rod. Probably some of you are familiar with this device. Basically it's a spinning rod. You put the rat on it and they run as long as they can because they have the instinct fear of height, which I can relate. And so they are scared to jump off so they start to, they try to stay on the rod as long as possible. And we can measure their endurance and motor function. So we administer different kind of ketones supplements to different strains of rodents, different age groups, and different administration methods, chronics of chronic acute. And what we found is that some ketones supplement in certain scenarios did improve the motor function. For example in this scenario with a four month old sprague dolly rat, ketones and ketones salt was the most beneficial. But it was interesting that in every scenario different ketones supplement was more beneficial. There is a publication on this so I encourage you to look into this if you are interested which scenario and which ketones supplement is the best. So while doing these experiments I also noticed two other interesting things. The first one that while I was measuring the ketones and the glucose levels of these animals, I found that the ketone level not only increased but their glucose level decreased. And not only the exercising rats but also the non-exercising rats had lower blood glucose in response to taking the ketones supplement, which can have important implications for people who would like to lower their blood glucose level. The other interesting thing that I noticed while doing these experiments, that some of the rats that were getting high dose of the ketones supplements, certain ketones supplements, they just looked down and they didn't care, they didn't have the fear and they jumped off the road. So the instinct disappeared. So I had the question, what happened with anxiety? I became curious and I started testing anxiety on the next batch of rats. So this is a generally accepted device to study anxiolegic drugs and similar things. It's called elevated plasmase and it's basically an elevated cross-shaped device which has to open and to close arms. Where the closed arms are surrounded by balls so when we put the rats in the middle, one rat at a time, if they are anxious they prefer to spend more time in the closed arms. So what we found is that again with certain ketones supplements the rats spend less time in the closed arms, they did walk less distance in the closed arms and the latency to enter to the closed arms was higher in response to taking the ketones supplements. So again different ketones supplements worked in different strains and different administration methods, different way. So not all of them get the same exact result but in general we can see that the ketones supplements were very efficient decreasing their anxiety. So after many more research experiments and publications now we know that exogenous ketones supplements have the therapeutic potential for different psychiatric disorders, not only anxiety but for example bipolar disorder and others because there are multiple mechanisms that are working synergistically to restore the brain energy metabolism, changes brain signaling and can reduce neuro inflammation. So the next experiment I was curious that okay we could improve motor function in normal rats without pathology but what happens with animals with pathology? So the next animal model that I used was glut 1 deficiency syndrome mice. If you are familiar with the disease in humans basically the glut 1 receptor is reduced in the brain so people have motor impairments and the ketogenic diet is actually very efficient to restore motor function in these people. So I was wondering we should test what happens if we administer ketones supplements to these rodents who showed the glut 1 deficiency syndrome instead of using the ketogenic diet. So I started feeding the mice with different ketones supplements for weeks and weeks. I measured their blood ketones and glucose every week and by the way their motor function improved but something more interesting came out of this study and I want to go in that direction. So when these animals got very stressed when I was taking their blood every week so I figured that maybe I will just put them in an anesthetic chamber so they fall asleep in a couple of seconds I take their blood and then they wake up in a couple of seconds so it's just less stressful for everybody. So while I was doing this I noticed that the mice that were getting the ketones supplement they just didn't want to go to sleep. I was just sitting there with my timer and the control animals they fell asleep in a couple of seconds and the ketones treated the animals that are still running around much later and they didn't want to go to sleep. So I started wondering and actually another study came out of this observation what happens with delaying the latency to anesthetic induction and it turns out that the animals that were fed the ketogenic diet or the animals that were getting ketones or ketones solved the ketones delayed the latency to anesthetic induction which is again proof of neuro-protection and it can potentially provide neuro-protection from other harmful gases too which again can be important for example people working around harmful gases such as firefighters. Again more details in the publications several other publications came out of this study too so if you are interested in more details I recommend to check it out. So during this time there were other projects ongoing in the lab relating to cancer. Why cancer? Otto Werber described first that cancer is a metabolic disease and the best demonstration is the fat scan that basically they are using to locate tumor because the cancer cells are over-consuming glucose and over-compete normal cells. A very good book about this cancer is a metabolic disease by Thomas Stiefer again if somebody wants to go into details I recommend to read this but also other studies showed that high glucose level also leads to poor outcome poor clinical prognosis and basically a blood glucose is correlated to tumor growth. So we also know that mitochondria is abnormal in cancer cells so the next question for me was what happened to cancer cells in the presence of ketones specifically what happens with cell surface transporters of the cancer cells and why is it important? Basically the GLUT1 transporter serves to transport glucose into the cell which is what we saw it's a major fuel for the cancer cells and the MCT receptors are used to transport lactate out which is again a fuel for cancer cells. So what I wanted to know is what happens to these specific receptors in response to ketone administration? So I studied different cancer cell lines so the first one is along carcinoma cell line from a white Caucasian male this is the control these four images and this is the treated image and the green labels the GLUT1 transporters in control you see the density is much higher and after beta-hydroxybutyrate administration the GLUT1 transporter density decreased. Also in another cell culture model which is a glioblastoma cell culture from a female 44 years old female subject the MCT receptors decreased too in response to BHP and also the GLUT1 receptors decreased this is the control very intense green and this is the treated less intense green less GLUT1 transporter was visible. This is a three-dimensional reconstruction of a cancer cell of a lung carcinoma. Again on the left this is the control you see a lot of green which is the MCT1 transporters and here is the BHP treated with much less green which is a much less GLUT1 transporter. These are the cross-sections of the cells and also just a quick video it's like slicing the cell like slicing the bread you can see inside and outside there's a lot of green a lot of transporters in the control cells and in the treated cells there's much less inside and outside of the cells. So the other interesting thing that I observe while doing the animal studies the cell studies and we soon started human studies too. I noticed that not all the results added up something was just not right and I started to look at the data what's going on and I noticed that there was some kind of concentration dependent effect but not in a way what you would expect. So next we looked at the GLUT1 transporter expression in response to BHP in cell cultures. We looked at in animal model the anxiolytic effect in response to ketone ester MCT and also we looked at insulin level in humans in response to ketone supplementation, ketone salt and ketone ester. And what we found is that at certain level this is another cancer cell line glioblastoma cancer cell line. Interestingly when it was a low concentration of beta hydroxybutyrate the GLUT1 transporter density decreased here but when we further increased the concentration the density of the GLUT1 transporter increased again. So we found that there must be some kind of compensatory mechanism when the ketone level was too high. Similarly in the animal study researching anxiety using another model which is a light-dark box test which basically if the animal spends more time in the light area it's less anxious and if the latency to entry to the dark is higher then it's less anxious. So basically we found again that the low concentration of ketone ester MCT decreased anxiety but higher concentration didn't work. Similar surprising results when a human consumed high dose of BHP ketone salt or high dose of ketone ester we found that in the case of ketone ester the insulin level increased which was not the case with ketone salts. These results kind of opened the question that maybe more is not always better. So we hear it very often that people are focusing on higher and higher ketone levels to achieve that but some results are showing that it might be not the best option at least not for every application. And this is probably the most exciting research for me, a research project that you probably see already on my poster. If not then I will quickly explain. So basically I got primary neurons which you can already buy in the store basically. And you played them in Petri dishes and you grow them and I scratched them to simulate brain injury and monitor them over 24 hours what happened to the damaged area by taking photos with a microscope every 15 minutes and then I created a time-lapse video. So what I found is that in the control Petri dish after 24 hours in the damaged area there was basically no cell. The damage remained about the same, nothing changed. But after 24 hours in the BHB treated Petri dishes the cells migrated into the damaged area and not only migrated into the damaged area but when we labeled synapses that shows new synapses for example here with red so these four is the control dish so basically here you can see barely any red or new synapses but in the BHB treated Petri dish there was a lot of new synapses forming around the damaged area. Also we used tubulin to label microtubulus which is green here. So in the control you see that the cells have microtubulus but in the BHB treated cultures the microtubulus density was higher and also we found them in the damaged area showing that the neurons actually started growing processes into the damaged area and this actually shows the same it's just a higher resolution image. This is the control, basically no red, no new synapses, a little bit more, a little bit of the tubulin but in the treated more red new synapses and more microtubulus was observed. And this is a video of the time lapse over the 24 hours of the control slides so basically you see that nothing really happened the damage is still there and the next video you will see the BHB treated Petri dish you can see that the cells are moving like crazy inside the damaged area growing processes and that some areas they are even almost closing the damage. So these results can have very important implication for people with brain injuries for example with traumatic brain injury or people who play sports or have a job then when they are exposed to concussions. So after these experiments two things happened. One I married my husband and I got fired because we were not allowed to work together anymore so I'm still trying to continue this research project. Well after that I started working with NASA and astronauts and DARPA so it's all fine but still these studies need to be finished by somebody. The other thing that happened is that during all these years I've seen enough about the potential of this molecule and especially this experiment convinced me that oh maybe I should be consuming these molecules. So I knew that at that time there was no commercially available ketone supplement that was optimally formulated that we would like to consume so I decided to create my own company, a dishes nutrition and USF actually enrolled me into a program that was established by the government to help scientists and academics to bring their invention into the market so people can actually benefit from it. So over a couple years I developed the product and you can get some outside if you feel like it, trying it. And during the last year since it was on the market I got a lot of good feedback from people with epilepsy, with migraine, with bipolar disorder that it really helped them that others didn't. So I feel that I could make a positive impact with this if you have any questions about the company or the products just find me after. And basically the lessons learned during these times based on the research that I did and also many hundreds of other researchers and hundreds of other research publications now we know that ketones can increase ATP energy production, can improve motor function, can improve mitochondrial function, influence gene expression, lower loss production, reduce neuro inflammation, can improve cognitive function, lower blood glucose and can help with neuro protection and neuro regeneration. The other lessons that I learned is that different ketones were good for different applications. So this is a research area that will need more attention because it's not general and we have to direct more research in the direction. The other lesson that we learned is that more is not always better as I mentioned before. And also it was very important lesson for me to keep an open mind when I'm looking at the experiment, what's happening, not just trying to prove something that I want to prove but actually observing what's happening with the cells, what's happening with the animals because maybe the new directions can lead to even more interesting results. The other lesson I learned is to have a backup plan when you marry your co-worker. But if anything negative happens just try to focus on the opportunities and not on the limitations because I think in this way you might get better results and can make a bigger impact than you originally planned. And with that I would like to thank many people who participated in this research project. My husband primarily Dominic Dagestino, many of his students, many of my students are a collaborator in Hungary, Zsolt Kovac and some of the companies who provided partial funding for accomplishing these projects such as Quest Nutrition, Keton Technologies, Office of Navy Research and the USF Foundation. And if you have any questions I'm happy to answer. Thank you. So the observation about delayed onset of anesthesia effect with isofluorine is fascinating because in the cardiovascular literature with heart surgery, isofluorine is known to produce pre-aschemic conditioning meaning if you expose someone before heart surgery to isofluorine compared to other anesthetics, the heart tolerates ischemia from being on pump or a total cardiac arrest. So can you relate that to the delayed onset with ketones? Yes, so that's very interesting. So actually we started talking to anesthesiologists, what do they think about all this and what is the application for their field to. So we started doing additional experiments on this subject and it's right now ongoing, mainly in Hungary to address some of these details. So I can't really answer the question but we are on it to find out more details, how is this actually working because like everybody was surprised. But this can be very important if people in ketosis are being put under anesthesia or different applications. Thank you. Hi doctors, thank you very much for your presentation. My question is, these appear to be mostly in vitro studies. So I'm kind of wondering about the applicability to obviously in humans which is in vivo. Are the ketone concentrations you're using similar to someone who is reasonably well keto-adapted, the same concentration as in the blood of someone? Like what concentrations are we talking about here? Yes, so we completely agree that all these needs to be moved to human research. So that's the next step. Some of the projects started, there are some clinical trials addressing these questions. Actually they are starting a clinical trial in a hospital in Norway using BHB for TBI patients. What is the ideal concentration and how does it relate to the cell culture studies? Again, it's a very difficult question to just give a generalized answer because every application, what we can see that every application is different and every ketone supplement is different and every person's metabolism is different. Like somebody who is following the ketogenic diet for years will probably respond differently than somebody who's completely new to the idea. So again, this is a very complex question and I wouldn't feel comfortable just giving a single word answer. Understood, thank you. Great talk, thank you. My understanding is that for ketones to be utilized in the citric acid cycle you need succinyl CoA and so when I saw your Glute 1 increase with higher ketone concentrations, my gut instinct is that the glucose would be needed to generate oxaloacetate to serve as a source of new succinyl CoA to deal with higher ketone concentrations and that you would be able to achieve optimal cellular utilization of the ketones if you had glucose or if you supplemented with oxaloacetate or glutamate or alpha ketoglutarate or citrate or anything that's upstream from succinyl CoA. I was wondering what you think about that because it seems like if that is the mechanism that there would be ways you could hack around that. Yeah, yeah, I think it's a good idea and probably it would be a more ideal combination that again we probably need to test because this was just one, a couple studies but we can probably optimize this further with compounds you mentioned and ideas like that. Yes, thank you. My questions are just a little more basic. Thank you, it's really interesting research and I followed some of the research over the years but the question I had is the rats that were given the supplemental ketone salts were they in a ketogenic state? It's kind of along the lines of what we heard earlier. Were they already in a ketogenic state in terms of how you prep them or was this ketones given within a regular glucose metabolism state? So trying to correlate that to a human population that's largely not ketogenic, that's one question. And then my second question has to do with long COVID research. COVID also apparently metabolizes glucose and I'm so just like cancer and I'm wondering if you've turned any attention towards that using ketogenic supplements. Yes, so going backwards I didn't do any COVID related research and I'm not comfortable addressing any of that idea because I just don't know I didn't do any of that research and the first question, what was it? The first question about the rats coming into the study that were given the ketone salts were they ketogenic to begin with or were they just on a mixed diet? So when I mentioned the treatment group as ketones or ketone salt or whatever ketone supplement they were on standard diet. So when I mentioned that ketogenic diet that group was on ketogenic diet but the others were on standard diet just taking the supplement. Thank you. Thank you for your talk here and the questions. There's a primal play session starting right now out meeting out in the lobby and in 20 minutes the next talk starts but Sheila would you be sticking around for some last-minute questions maybe? Yeah, sure. Okay, one, two more questions. Yeah, mine's quite a bit a lot. Okay, really, really fascinating and so similar to what Ben was saying this is all in vitro. Do we have any studies out there yet that are showing even if they're observational only some sort of effect on humans like how do we know like you've got a supplement company great but do we have any kind of evidence that it actually translates into humans yet? Yeah, there are many studies on humans too. I didn't specifically do that but there are many studies in the literature. You can find them on our website that we reference them or you can just do a Google research. So yes, during the last couple years more and more research started to focus on specific supplements or just in general inducing ketosis with either the diet or with the supplements but there are studies out with humans. Okay, excellent and so we are seeing some effect on humans on let's say anxiety. Yes, so not all the applications that I mentioned here but some applications, yes. Okay, great, thanks. Excellent talk and really impressive series of experiments that you presented. I'm curious with a lot of the rat experiments or the rodent experiments as well as with the tissue was that extracted from male rats or were you controlling for sex differences and looking in female rats as well and if not would you expect that there would be differences in some of the findings you observed? Yes, that's a very good question. Thank you. So these experiments that I presented here these are all male rats we started also some experiments that are gender specific so we actually have a paper that I didn't mention here looking at ketone and glucose level in female and male rats and it's different and also different applications for example anxiety we are having experiments right now in Hungary running some studies on anxiety and the female and the male rats respond differently so stay tuned it will be published really soon. Looking forward to it, thank you. Thank you. Let's thank again Dr. Adi Dagestino for sharing her work with us. Thank you. And let's please meet back here.