Dominic D’Agostino, Ph.D. on Modified Atkins Diet, Keto-Adaptation, Ketosis & More

[Rhonda]: Hello, everyone, I’m sitting herewith Dr. Dominic D’Agostino in Tampa, Florida. Dominic is an assistant professor at the Universityof South Florida where his primary research focus is on metabolic therapies, particularlynutritional ketosis, ketone supplementation, and how they affect a wide variety of pathologicalconditions ranging from neurological disorders like ALS to epilepsy to muscle wasting andcancer. So very, very interesting stuff that Dominic’sdoing here in Florida. He showed me some of his really cool equipment,which is super-exciting and very interesting.Some things I hadn’t seen before. So welcome, Dominic. [Dom]: Thanks for having me. [Rhonda]: Yeah, thanks for inviting us toyour lab. [Dom]: Great to have you here in my lab, yeah. [Rhonda]: Super-cool. [Dom]: Thanks. [Rhonda]: So tell us a little bit about your…someof your more recent interests and research where you’re, sort of, been, what have youbeen looking at. [Dom]: Okay, so more recently, I’d say withinthe last three to five years, and it took a while to develop them, but as you know we’reworking on ketone supplementation. And the idea is to, kind of, mimic the therapeuticeffects and performance-enhancing effects of the ketogenic diet. So nutritional ketosis achieved through adietary means with a low-carbohydrate, high-fat diet has therapeutic effects for a broad rangeof neurological disorders, in particular, seizures. And you can make the argument that prettymuch every neurological disorder is some way linked to a metabolic dysregulation. And we’re interested in understanding hownutritional ketosis may help to preserve and, sort of, stabilize brain energy metabolismto metabolically manage these seizure disorders.So in the process of understanding and studyingthe ketogenic diet, we are developing a broad range of ketone supplements which can includeketone esters and ketone mineral salts, which where we combine a ketone body, beta-hydroxybutyrate,to an essential electrolyte and create a wide variety of salts. And we’re working…a lot of the work nowis focused on formulating these to make them tolerable, to make them palatable, and tounderstand their therapeutic potency for different types of disorders. [Rhonda]: Yeah, so you mentioned that at theroot, or at the heart of a lot of these neurological disorders is a metabolic dysfunction and how,first of all, when we talk about nutritional ketosis, to me, it’s so, it’s such a broadway of describing. [Dom]: Very broad. [Rhonda]: What is nutritional ketosis? Like, and how do you achieve it? [Dom]: So nutritional ketosis could be, it’sdefined…they, kind of, have the same definition in my mind. It’s achieving and sustaining a level of bloodketones. And I think when people say they’ve done theketogenic diet, they did that it didn’t work for them or it did work for them, I wouldask the question, “Well, did you measure blood ketones? Did you confirm, in fact, that you were ableto achieve a state of ketosis?” Defined as an elevation of blood ketones above0.5 millimolar at the very least.Ideally, you want to stay between one andthree millimolar of ketones. And when you’ve achieved that state… [Rhonda]: You’re talking about blood ketones? [Dom]: I’m talking about blood ketones, yeah,which is, kind of, the gold standard. And this can be measured. There’s a number of different devices outthere that measure blood ketones. And when that…the state of nutritional ketosisis achieved, you’re also, not only not only that biomarker is, kind of, there and we havetechnologies to measure it, but it would also be important to measure your blood glucoseand maybe insulin levels, too. The suppression of the hormone insulin driveshepatic ketogenesis and drives the body’s ability to make ketones. And that has therapeutic implications fortype 2 diabetes, obviously. So my definition of nutritional ketosis wouldbe an elevation of blood ketones and the, kind of, the difficulty in prescribing thator telling someone to do it is that the way to implement that is, kind of, similar witheveryone, but everyone responds differently depending on where you’re coming from.So if you have an obese subject that’s type2 diabetic, it’s going to be different than an athlete. And women, there’s some differences betweenwomen and men, I think. So defining nutritional ketosis is relativelyeasy with an elevation of blood ketones, but implementing it and being able to, for theindividual to commit to it and have that, kind of, ability to control their diet, whichis very linked to lifestyle, has been, sort of, a difficult thing to do. And that’s where ketone supplementation, kindof, comes in and can allow someone to rapidly achieve nutritional ketosis and sustain itand perhaps get many of the therapeutic benefits that we’re just finding out now. We know that ketones are more than just ametabolite. They are more than just an energy metabolitethat the brain can use, but they are metabolites, especially beta-hydroxybutyrate, are verypowerful signaling molecules.And we’re just beginning to understand, sortof, the therapeutic effects of these metabolites as signaling molecules and that’s a big thrustof our lab right now. [Rhonda]: Oh, you’re interested at lookingat this and the effects… [Dom]: Yes. [Rhonda]: Interesting, I’m familiar with someof Eric Verdin’s work at Gladstone and how he’s… The science… [Dom]: He’s a pioneer in… [Rhonda]: I’m very interested in beta-hydroxybutyrateas not only as a source of energy for mitochondria.So being able to be converted into a thermodynamicallyfavorable source of energy, but also the fact that it’s able to change cell signaling inthe brain and it’s able to turn on genes that are involved in dealing with stress better,some of these genes are involved in longevity, FOXO3 for one. [Dom]: Absolutely. [Rhonda]: So it’s all very interesting. But something that I…like, in my mind thatI’m not exactly certain about is that nutritional ketosis.So eating a high-fat diet and, of course,there’s, okay, what types of fat are you eating? Are you eating more polyunsaturated, you’reeating more saturated? You know, how much protein are you eating? What types of carbohydrates? Are you getting fiber? I mean, there’s so many diets, very complicated. But there is no doubt that there are interestingtherapeutic effects from nutritional ketosis. I am interested in the the actual end product,which is ketone bodies and these signaling molecules like beta-hydroxybutyrate beingable to get those maybe even without having to eat a high-fat diet, you can get them fromfasting, from intense exercise, right? Also, you can achieve…[Dom]: Post-exercise ketosis. [Rhonda]: Post exercise. [Dom]: Absolutely. [Rhonda]: So, I guess, the, kind of, the questionI had for you with nutritional ketosis besides having to define it was, what do people eatto obtain that? How do you get blood ketone levels betweenone and three millimolar? Like, what do you have to eat? What do you not have to eat? [Dom]: Okay, that is a good question and itvaries depending on who you talk to and what’s optimal.I think a good way to approach it would tobe describe what has been used classically and how the diet has evolved over the lasttwo to three decades, I think, with some of the work with Eric Kossoff at Johns Hopkinshas, kind of, advanced the idea of using a modified Atkins diet or modified ketogenicdiet. And I can, kind of, talk a little bit aboutstrategies that I use, I think, and others that I know use to achieve that state andsustain it for optimal performance in health, I think. So in taking a step back, the classical ketogenicdiet would be like a 4:1 or 3:1 diet, and that’s, kind of, the ratios of the macronutrients. Four being fat and one being a combinationof carbohydrates and protein together. So a pretty protein-restricted diet. And the ketogenic diet is not a high-proteindiet. It’s actually moderate to low protein diet. And most people don’t understand that, theythink, especially in the fitness community, if they go on a ketogenic diet…if they saythey’ve been on a ketogenic diet, what they will describe to me would be a very low-carbohydrate,high-protein, moderate-fat diet, but the ketogenic diet as it is used classically for drug-resistantepilepsy, the original was, like, 90% fat, like 85% to 90% fat and maybe about 10% protein,typically, and 8% to 10% protein, and a very minimal amount of carbohydrates, and it washeavily based upon the used of dairy fat.So dairy was the primary, kind of, vehicle,yeah, to get calories into you. And we know that the use of dairy especiallyin some people, dairy protein in particular, but even sometimes dairy fat, too, can havenegative consequences for some people. So there’s a wide variety of, kind of, ketogenicdiets out there and they, the ketogenic diet has been defined by this ratio of macronutrients. But what we’re learning now is that it’s morethan just macronutrients to optimize the diet, especially for individuals. The sources of the macronutrients, the fattyacid profile, the type of protein, it allows for some amount of carbohydrates, and thetypes and quality of the carbohydrates as it relates to gut health and gut microbiome,I think, is really important. I think it’s really important to optimizethe diversity of carbohydrate sources in the form of raw vegetables, and I think that itcan optimize the diversity of the gut microbiome, too. I think those two are linked, the diversityof the foods in your diet and the diversity of your gut microbiome.I’ve seen that just through feedback. It’s not really studied, but it’s somethingeveryone knows and it should be studied. So the classical ketogenic diet is very strictto follow. There’s a few studies showing that it caninfluence a lipid profile in a negative way, meaning a high elevation of LDL. And in kids, I think, that follow the diet,they had a high level of triglyceride. There was one study that’s often referencedin regard to the ketogenic diet being atherogenic the triglycerides are really high in someof the kids. [Rhonda]: It’s probably a very complex gene-nutrientinteraction, as well. [Dom]: Absolutely, yeah. [Rhonda]: There’s some, I’m not sure if you’refamiliar with any of this work.It’s something I’m getting into recently isthis nutrigenomic field and particularly, there is PPAR-gamma and PPAR-alpha, whichis important for ketosis and polymorphisms that are associated with not being able torespond well, so it’s very likely that some of these people that don’t respond well ormay have that polymorphism. [Dom]: Yeah, undoubtedly, because there aresome people that really have a negative effect, but I would it’s, kind of, few and far between…maybeas high as 20% in some circles, but generally speaking, if you have a dietitian that’s prettysavvy and has worked with enough people, I really emphasize experience because there’sno substitute for experience.So you could know as much…yeah, you couldknow the biochemistry behind nutritional ketosis, all the pathways, whatever, but working witha dietitian, I always stress that in people that want to try the ketogenic diet to startoff with a good dietitian. They can tailor and tweak the diet for yourspecific needs and I find that, for me, personally, a diet that’s low in dairy is really essentialbecause I just don’t do good with dairy, and nuts have been a staple for some people forthe ketogenic diet and I have, like, a mild nut allergy. So that’s another food that I’ve, kind of,had to eliminate. But despite that, I have been following whatI call a modified ketogenic diet and I think it’s pretty similar to the modified Atkinsdiet that Eric Kossoff has been using at Johns Hopkins.He’s at Johns Hopkins, mainly a neurologist,but has a team of dietitians working with him and has done an incredible job advancing,sort of, the use of the ketogenic diet. You know, he follows in the steps of JohnFreeman, who was really a pioneer in getting the neurology world to recognize a ketogenicdiet as a viable metabolic therapy for drug-resistant epilepsy. And he did a lot of work with The CharlieFoundation and worked with them to help promote awareness of the diet, education of the diet.So some work that Eric Kossoff has been doingover the years and publishing on is showing that the modified Atkins diet has much ofthe therapeutic potency of the strict classical 4:1 ketogenic diet. So instead of 90% fat, the modified Atkinsdiet is roughly 65% to maybe 70% fat which is, kind of, what I follow now, and about20% to 30% protein, with the balance being still very low on carbohydrates, like no morethan 5% or 10% carbohydrates.But it’s more liberal in the use of protein. And it’s also, advocates the use of medium-chainfatty acids, can be incorporated into that. So by following a diet that’s more liberalwith the protein and less not restrictive, and less in of a need of a such a high-fatcontent, I’ve been able to maintain a moderate state of ketosis as do many of the therapiesor many of the patients that are benefiting from this therapy for a wide range of disorders. So I think that makes the diet more accessibleto people, and I think the biggest hurdle now is just compliance. So people know the benefits of the diet, someof them are a little concerned with the side effects of the diet as far as lipid profilesbeing altered. [Rhonda]: What about gut? [Dom]: The gut, yeah. [Rhonda]: Have you been looking…how longhave you been doing this modified ketogenic diet? [Dom]: It, sort of, evolved from 2008 to ’09,I started experimenting with it because that’s when I got interested in using… [Rhonda]: And you’ve continued on ever sincethen? [Dom]: Yeah, I’ve never really gotten outof ketosis.[Rhonda]: It’s quite a long time. That’s quite a long time. [Dom]: Yeah. If I did, I may have reverted back to, like,a low-carb Paleo which is maybe up to 40% to 50% fat or protein on some days. So I’ve been a little more liberal on theprotein at times, but yeah, I’ve been following it for a long time. And I do blood work really consistent. [Rhonda]: Well, the reason I ask is because,it’s the one, like I mentioned, I’m very interested in nutritional ketosis, which is why I’m excitedto talk to you because you know so much more about it.And I’m trying to, like, get to the bot-,…there’sthis conflict in my head with eating a lot of fat and its effects on the gut becauseI know a lot of my gut researchers, a lot of my friends are, kind of, looking at guthealth and one of the main things that you do induce endotoxin, which is released frombacterial cell membranes. I remember it’s one of main things neededto induce gut permeability is if you have a high-fat diet. You feed mice, you feed…this is all donein animals, of course, which is… There’s lots of problems with that, but… [Dom]: What’s the typical fat source for that? Is it lard or is it…? [Rhonda]: A variety of different fat sources. There’s lard, there’s corn oil. I mean, so there’s there’s a variety of differentfat sources, but fat itself in order to be digested, you have make these bile acids likedeoxycholic acid, which which causes endotoxin release.It also acts as a surfactant. I mean, it’s like a detergent. So, I’m not convinced that it’s not not healthy,but I’m not…I’m, sort of, trying to get to the…there’s a disconnect in the literaturebecause there’s so much information out there showing the benefits of a ketogenic diet,nutritional ketosis. [Dom]: Let me ask this question real quick. So when endotoxin is released from these bileacids, so there would be a predictable, a characteristic cytokine profile that wouldreflect that, right? [Rhonda]: Yes, so there’s a cytokine profileand you also measure endotoxin in the blood, which is a very tricky thing to do becausethere’s a lot of false positives and I know someone who’s trying to develop an assay tomake it actual-, because he’s very OCD about it.It’s not out there for clinical use yet becauseof that reason, and even doing it in animal studies, there’s a lot of researchers thatdon’t really do it right, measuring endotoxin. [Dom]: Is there any benefit to endotoxin? So when I go exercise right, and you measuremy blood and we look at reactive oxygen species or inflammatory mediators, you could lookat the blood and say, “Don’t do that, this is not a good thing to do.” Whereas if you have periodic spikes maybeand endotoxin, is it stimulating a hormetic effect where it’s enhancing my resilienceor resistance to toxins, do we know that? [Rhonda]: So endotoxin, I would say basedon everything that I have known and researched and from my interactions with people thathave been doing this research. It’s not like reactive oxygen species wherethere’s it’s a potent signaling molecule that has this hormetic effect, increases mitochondriabiogenesis, it increases all these genes involved in dealing with stress there’s lot of benefits. You know, endotoxin release from the gut,one, it causes more VLDL production because VLDL sucks soaks it up.So that’s part of the reason why inflammationis also correlated with an increase in LDL number. It also binds to ApoB, it binds to the part,it binds to where the LDL receptors bind so that LDL can’t be recycled as well. So it, kind of, prevents. There’s a lot of bad things about endotoxinbeing released. Now I don’t know, maybe there is some, sortof, slight benefit from endotoxin being released.You know, I don’t want to get too into theendotoxin world. I’m just saying there is, in my…I’m tryingto, like, understand in my mind how…. [Dom]: So you might be a little biased becausesome of the high-fat diet work has endotoxin. I’m a little bit, I need to get educated,I mean, I understand endotoxin from, kind of, like a basic science point of view. [Rhonda]: Right, like, “sepsis, bad.” [Dom]: Yeah, from “sepsis, bad,” but I doknow when your body is challenged, even things like radiation.I mean, there are some things…you know,I don’t know of any case where an auto antibody is a good thing, but I know most of the thingsthat are bad out there do have some benefits, it’s the level it’s the level. [Rhonda]: Absolutely, dose is very important. [Dom]: And the phenotype’s ability to adaptto that chemical, that stimulus is really important. Like, old people don’t adapt to older phenotypesdon’t adapt to an oxidative stress stimulus as robust. So I would be interested, and there’s waysto simulate endotoxin, right? Experimental models and that’s something wecan do, challenge, perhaps run an experiment where we have animals on different diets wherewe can challenge them and look at inflammatory cytokines.We’re doing a lot of that work now. [Rhonda]: And gut health in general, gut permeability. I would love to see some ketogenic researchgo in that direction because there’s so many unanswered questions in my mind. And there’s a lot of bad data out there, likeso from the high-fat diet and the effects on the gut because when you look at controlleddiets and the high-fat diet, well, the controlled diets have, like, 50% more fiber, and it’slike, “Well, that’s not really the same thing if you’re just changing fat.” So there’s, you have sift through all sortsof crap, and it’s, like, there’s lots of data you just have to throw out, but still at theend of the day, I’m uncertain and I’m weary about me, eating a high-fat diet. Not not mention the fact that I have certaingene polymorphisms that may not be as compatible, but because of that, because of gut health,that’s my major, the limiting factor for me is gut health and I like to see more researchon ketogenic diets and gut health, like, that’s something that I think is important.[Dom]: How to optimize it. [Rhonda]: Yeah, or just what the effects are,like maybe they’re for one, we’re starving out probably a lot of the pathogenic sugar-eatingbacteria. So there’s probably some good things goingon. And then there’s, well, what effect does medium-chaintriglycerides has? Is it the same as long-chain fats? Maybe not. So maybe…so there is different types offat there’s studies that had been coming out very recently showing the effects of polyunsaturatedfat on the gut biome, and how it has a positive effect. So maybe if you eat certain types of fat,more of these types of fat versus the others.There’s lot of things out there to be explored. [Dom]: Undoubtedly shifts it, yeah. It shifts it. I mean, your gut microbiome has an appetitefor whatever you’re feeding it and that probably influences profoundly your own appetite. So I think if you have sugar-eating gut microbes,you’re probably going to be craving sugar if you go a few hours without having sugar. And I think I would be very interested innot only, like, shifting someone from a high-carb diet to two or three different types of ketogenicdiets, I think, would be important with specific fatty acid compositions and fiber compositions.And I think it’s the diversity of the fiberthat’s incorporated. [Rhonda]: Diversity, yes, it is very important. There have been there’s lots of differenttypes of bacteria and they’re eating different precursors to generate a lot of these othersignaling molecules, short chain fatty acids and things like that that are regulating yourimmune system, that are regulating literally hematopoiesis, Tregs, natural killer cellslots of…and this is a whole blooming field of research. That’s also another reason I’ve been a littlehesitant to experiment with this because I’m so, fiber is so important for me, fiber fromvegetables. You know, I’m not adverse to fiber from fruits,so fiber from fruits, legumes, beans, like, I like to get a lot of fiber in my diet, andso I don’t know if that, is that compatible with it? [Dom]: I get more fiber on a ketogenic diet.You know, and we’re talking about a well-formulatedketogenic diet, as Jeff Volek likes to… And because that is really important becauseyou talk to people that you eat ketogenic diets and it’s all over the board. But I think a well-formulated ketogenic dietwould have an abundance of fiber sources, everything from green vegetables, of course,but would include a salad, and I think it’s really important from a gut microbiome perspectiveto get in raw vegetables, I think, from what I’ve known and a half-dozen individuals thatI talk to that are, kind of experts in this field.They think that that has a pretty profoundeffect, and I have always done that and I would say my gut health has not… It may be due to what I eliminated in my diet. I grew up in an Italian family eating a lotof pasta, bread, so they were the staple foods, and I gravitated toward a Paleo diet mid-90s,early 2000s and then the ketogenic diet, and I have never had better gut health than whenon a ketogenic diet, but my diet is, like I said, more of a modified Atkins and hasa pretty liberal amount of vegetables in it.And I think the benefit to including the vegetables,they’re just carriers for the fat and they also slow protein digestion, which helps minimizethe insulin spike that you can get from protein and helps keep me in ketosis. But, I mean, getting back to your questionabout the ketogenic diet or high-fat diet influencing endotoxin or factors, I wouldthink that would show up in the literature that if some, and maybe it does, but it usually,a high-fat diet is in the context of a high-carbohydrate diet, too. So that’s what we don’t know when we talkabout LDL particles being elevated, like, even skyrocketed, LDL, little peak. That is only understood in the context ofa high-fat diet, which also includes also sugar like a western diet. So we don’t yet understand lipidomics andthe shifts in lipid profile of the ketogenic diet, LDL(p), specifically, we don’t understandit in the context of ketoadaptation.[Rhonda]: Okay, what’s that? [Dom]: Ketoadaptation, I would, it’s, kindof, a term that Jeff Volek and Stephen Phinney coined, but I think it’s very descriptivein my mind as a physiological process when your body has adapted to using fatty acidsand ketones for fuel where you’ve biochemically and physiologically shifted your metabolismfrom burning glucose as the primary fuel to burning glucose and also equal or maybe more,in some cases, ketone bodies primarily from your central nervous system. So when that happens, that’s acutely, youget an elevation of blood ketones and over time, what you do is get an upregulation ofthe transport mechanism so your ability to make ketones, utilize ketones, and metabolizeketones in the cell is dramatically increased as is the oxidative capacity of your cells.[Rhonda]: After you start making more ketones. [Dom]: After, yeah. It’s, sort of, driven by being in a stateof ketosis enhances fat oxidation over time. So when we say ketoadaptation, we should probablysay keto and fat adaptation. So there’s studies out there that show ourmetabolic physiology changes pretty profoundly from eating this. We basically, we burn what we eat, right? So with a high-carbohydrate diet, we are prettymuch burning that as fuel and our bodies can adapt remarkably well to burning a macronutrientprofile that’s reflective of the ketogenic diet.And when we do that, a lot of really remarkable,beneficial metabolic processes happen including mitochondrial biogenesis, reduction of ROS,reduction of inflammation, a reduction of specific inflammatory cytokines that may beassociated with with age-related chronic diseases are reduced. [Rhonda]: And so reduction of ROS, becauseI would, no, I guess some of Richard Veech’s, have put some ideas out there about the… [Dom]: Ketones, direct effect, yeah, on that,too. [Rhonda]: Yeah, it’s so funny because in mymind I always think about like, well, if you’re inducing your mitochondria to work more, you’regoing to make more reactive oxygen species, right, and that’s, sort of, what I think wouldbe a driver of killing abnormal cells, cancer cells that are primed to die. They’re expressing way more pro-apoptoticproteins they’ve countered it with anti-apoptotics. All they need is a little push, a little reactiveoxygen species pushed to death. [Dom]: And that’s increased initially. [Rhonda]: It is. [Dom]: So yeah, it actually activates likea Nrf2. So when someone gets on a ketogenic diet,it’s a stress to the body and you…[Rhonda]: Yeah, so you are…you aren’t makingmore, because if you are causing your mitochondria to only work, right, that’s the only way youcan make energy, then you’d think that you have to be making reactive oxygen species. At least if you’re looking in the contextof a cancer cell, which just would be glycolytic and not using the mitochondria at all, right? Then definitely there would be a much more,an increase in ROS. So ketone, so you’re saying that it can suppressROS.Is that through some of what Richard Veechhas put out there in the semi-ubiquinone, how it’s like… [Dom]: Oxidize Q, makes it less available. [Rhonda]: Yeah, right, because that’s themost… [Dom]: If Q is oxidized, yeah, so. [Rhonda]: So Q being ubiquinone. [Dom]: Yeah, ubiquinone. If ubiquinone is oxidized, which is achievedwith our beta-hydroxybutyrate metabolism, if that’s oxidized, then you have less availabilityfor that electron to react with molecular oxygen in the metabolic pathway. So you would produce less superoxide anion,which is your precursor to more reactive oxygen species. And that’s been shown elegantly in a numberof models including the cardiac model, which he did, the Langenhorn [SP] model, the perfusedheart preparation showing that you get a greater hydrolic efficiency of the heart in the presenceof ketones. You know, with a given amount of oxygen, youcan generate proportionally more ATP, energy currency. [Rhonda]: Is there another way that ketonesalso suppress ROS? Or is that through the hormetic effect ofactivating mitochondria, then increasing Nrf2 and things like that? [Dom]: Yeah, that would be a secondary effect. I would say from a acute point of view, assimply as a metabolic fuel through the mitochondrial efficiency is greater.So you have a greater mitochondrial membranepotential, a greater driving force for ATP synthase to make ATP. So it energizes the mitochondria in a waythat would be expected from a metabolic fuel that’s, sort of, superior from a bioenergeticpoint of view. So you have a greater capacity to generateATP for a given amount of oxygen that’s available. So with that occurring, the metabolic efficiencyof the cell would be, sort of, preserved, you’re using less oxygen to make the sameamount of ATP, less reactive oxygen species.And, of course, if you’re shifting away fromglycolysis and shifting towards oxidative phosphorylation… [Rhonda]: So in the context of a cancer cell. [Dom]: In the context of, yeah, any kind ofcell, like our tissue, really, skeletal muscle or cancer cell, yeah, you are forcing thebody in a way and to stress, initially, to upregulate mitochondrial machinery, really,and more mitochondria will start budding off and creating mitochondrial biogenesis. [Rhonda]: So it increases mitochondrial biogenesis. [Dom]: Yeah, the number of mitochondria. Then, the proteins that are associated withthe electron transport chain, those proteins are upregulated.[Rhonda]: That’s very interesting. It acts very similar to lactate. I don’t know if you’re familiar with any of,like, the brain work, and George Brooks, and lactate. [Dom]: Yeah. I…very interested in lactate as a graduatestudent. [Rhonda]: Because it does the same thing. Goes to the same transporter, right? [Dom]: Yeah, at MCT. I studied lactate as, sort of, an undergradand graduate student, like, I am studying ketones now as an alternative fuel. [Rhonda]: Oh, really? We’re you looking at it in the brain? [Dom]: Yeah, during hypoxia.So I studied the neural control of autonomicregulation, so brain hypoxia and what our brains do under hypoxia. And lactate is a big player in preservingbrain function, viability, health, and I studied lactate as an alternative energy source underhypoxia. And now, I think of ketones as, like, thealternative energy source when your brain is under normal physiology. And I think there are some uses for lactate,too, as a fuel. I think when we exercise we’re creating lactateand we feel good, it’s not really talked about.It’s something that I want to study and maybetalk about a little bit more is that the lactate…you’re also sending, not only are you sending ketonesto your brain, but you’re sending lactate to your brain, and I think that’s maybe nottalked about that much, but there’s a potential out there for lactate supplements. [Rhonda]: George Brooks in UC Berkeley, he’s…Idon’t know if you’re familiar with any of his work. [Dom]: A little bit, yeah, peripherally. [Rhonda]: He pioneered the whole lactate shuttletheory, but he’s been looking at the effects of lactate generated during exercise, forexample, when you’re forcing your muscle cells to work harder and you’re making more lactate,it gets over, it crosses over the blood-brain barrier gets into neurons.So neurons themselves actually use lactategenerated from astrocytes. So they are using, I mean, neurons actuallyusing lactate. It’s also a thermodynamically and energeticallyfavorable source of energy much like ketones. And so neurons like doing that because, one,it’s easier, and two, because glucose can then be freed up to be shunted into the pentosephosphate pathway, which can be used to generate NADPH, which is important for glutathionerecycling. [Dom]: Antioxidant capacity, yeah. [Rhonda]: Right, which makes sense why… Probably, I think there’s a lot of parallelsbetween ketone bodies and ketone supplements and how they’re not only being used as a preferentialsource of energy in the brain. Do you know anything about this, about, like,how frees up glucose then to be used for other… [Dom]: Glucose sparing. [Rhonda]: …metabolic pathways? Yeah, glucose sparing. [Dom]: Yeah, it’s thought that some of thework by Steven Cunnane, I think, is shedding light on this, too.[Rhonda]: Where is he at? [Dom]: He’s in Canada. I forget the institute that he’s at. Joe LaManna has done some similar work andhe’s at Case Western and looked at the, kind of, the interaction of what does the brainprefer…what’s the preferred fuel source for the brain. I get this question a lot. I think it depends on, you know…I don’tknow if it’s right to say that the brain always prefers ketones. [Rhonda]: The brain cell we’re talking about,for one. The neuron, astrocytes. [Dom]: Neurons and astrocytes. But, yeah, I guess, I mean, maybe we’re definitelybiased towards understanding neurons relative to astrocytes. [Rhonda]: I think most people that ask methat question are….[Dom]: Astrocytes are fascinating. I think we need to study that more. But I think in the context of, like, agingand a context of traumatic brain injury or pathology, I think the brain will really preferto use ketones because…or in the context of some kind of stress hypoglycemia or somethinglike that, I think the brain will also prefer to use ketones. [Rhonda]: Yeah, so why do you think that is? [Dom]: Well, I think… [Rhonda]: I might have my own reasons why. [Dom]: There’s a whole host of reasons orthings that can cause impaired brain energy, brain glucose metabolism and that could beinternalization of the GLUT3 transporter, which occurs…it’s kind of linked to Alzheimer’spathology. [Rhonda]: GLUT… [Dom]: GLUT3, yeah. And there’s a couple of key enzymes that areeither deficient or not active like they should be, pyruvate dehydrogenase complex is deficient. [Rhonda]: So wait, are these in…are theGLUT3 changes in astrocytes or in neurons? [Dom]: Primarily in neurons.[Rhonda]: Neurons, okay. [Dom]: So the glucose transporter in neurons. [Rhonda]: So if neurons are using lactateprimarily…the astrocytes are actually what’s… [Dom]: Shuttling a lot. [Rhonda]: The astrocytes are using glucoseand that’s why the brain needs glucose and they’re producing the lactate. The neurons are using the lactate becauseit’s getting shunted and converted into pyruvate. [Dom]: And the contribution of that is notcompletely known, but it’s pretty significant contribution during hypoxia, I would think. [Rhonda]: There’s been studies out there showingthat not just during hypoxia, but actually just under normal physiology that… [Dom]: It’s a key player. [Rhonda]: It is, yeah, the neurons are gettinglactate continually from astrocytes which are generating it, but that the astrocytesbecome aberrant during…for whatever reason, it’s not known, Alzheimer’s leaves traumaticbrain injury, so they can’t make that lactate and neurons have to start using the glucose,which then is, but I mean, that’s just one.[Dom]: Yeah, I’ve heard about that, I haven’tlooked into that. Yeah, so there’s some key enzymes then thatpeople look at not only just the lactate levels… [Rhonda]: I think that…I have no idea whatthey’re looking at, but for the pyruvate dehydrogenase complex, you’re saying that’s also aberrantin some of these disease states because then, you wouldn’t be able to use the lactate becauseyou couldn’t convert it into pyruvate. So anyways, it’s all kind of, I totally interruptedyou, though, but you were talking about… What were you talking about, the aberrantenzymes, like GLUT3 and pyruvate dehydrogenase, and how these are aberrant in different neurologicaldisorders. [Dom]: Yeah. So I think, I think, well, maybe the questionwe’re trying to get at is the contribution of ketones as a brain energy source and especiallyin, sort of, in academia and circles where I teach, it’s not even, still not really known,it’s not really accepted or understood that much.But I think the capacity, the metabolic flexibilityof the brain to be able to use ketones, we can exploit that and we do that with nutritionalketosis, and it’s altering brain energy metabolism and the neuropharmacology of the brain inways we don’t completely understand now, meaning the neurotransmitters in the brain. So I could draw off GABA. So you have more GABA, the GABA-to-glutamateratio is shifted in favor of higher GABA. So there’s a higher GABA-to-glutamate ratiowhen one is on the ketogenic diet. [Rhonda]: In people that are not having…innormal people, as well? Just, like, in general? [Dom]: Yeah. [Rhonda]: Interesting. [Dom]: It’s thought that, and there’s differentreasons for that. There’s glutamic acid decarboxylase is anenzyme. [Rhonda]: Is that how it helps with epilepsy? [Dom]: That’s part of it. I think there’s about 20 or more things goingon if you look at all the signaling pathways [crosstalk 00:39:37] actually what happens,yeah, with metabolism, but I think a key player in that is an elevation of GABA to glutamate. And we need glutamate to make GABA, right,but the enzyme is elevated and the pathways are shift in favor of more glutamate to GABA,which has a stabilizing effect on your cell membrane and neural interactivity, in general.[Rhonda]: How readily do ketones cross theblood-brain barrier? [Dom]: It’s thought up to five millimolar,maybe six, seven millimolar, they can readily cross the blood-brain barrier. [Rhonda]: You have to get that high beforethey start crossing? [Dom]: No they start impeding once you getlevels up that high. It hasn’t really been studied in-depth. We did brain metabolomics to show that whenwe looked at the ketone levels in brain tissue and ketone levels in the blood, and there’sa high correlation there. Interestingly, we had a diet, too, that washigh in medium-chain fatty acids. And although I heard the medium-chain fattyacids could readily cross the blood-brain barrier long-chain fatty acids typically don’t,short-chain fatty acids sort of do. But we found very high levels of medium-chainfatty acids indicative of them and these were normal, healthy animals. So when you take medium-chain fatty acids,they are readily from the looks of our metabolomic data, just readily crossing the blood-brainbarrier and capable of being used as fuel…[Rhonda]: That’s fascinating, I had no idea. [Dom]: …which is very, yeah, it’s reallyinteresting. I heard about that, but when you see the datait’s at really high levels. [Rhonda]: So when they’re in the brain, thenhow are they…so then what happens? So they’re used… [Dom]: Yeah, their medium-chain fatty acidscan be oxidized just like fatty acids for fuel, yeah… [Rhonda]: Wow, interesting. [Dom]: ..uh-huh… oxidative, but ketonesare also very high in the brain and to really capture and understand it, you have to, sortof, capture the brain tissue really quick and freeze it and then do an analysis becausethe brain’s using it as fuel after you take it, your brain’s, kind of, still alive.Sort of, the step that I was showing you inthe lab where we can take the brain out and cut it like a piece of bread and then recordfrom it. So that brain is still active. So it, sort of, uses the metabolites. So to get a very clear snapshot of the differencein tissue versus blood is a little tricky as is getting ATP levels in the brain.It’s a little tricky. We’re working on that to be able to do that. Another question which I think will involveradio tracers is actually to do, like, a ketone PET scan where we can look specifically atthe regional distribution of ketones and figuring out what areas of the brain may be preferentiallyshuttling them and using them as fuel and how that may change under different pathologies,how that may change under oxygen deprivation, glucose deprivation, high oxygen, as I studyoxygen toxicity. [Rhonda]: Please look at genetic states likeApoE4 because that’s one thing… [Dom]: Yeah, that’s a biggie. [Rhonda]: Yeah, I’m very interested in someof this work you’re talking about in ketone supplements helping with neurological disorderslike Alzheimer’s. It’s been shown that that is dependent onApoE status, and that just, it’s disappointing to me because I really want to understandwhy and I just… Do you have any idea? [Dom]: Well, so you’re referring to the Hendersonpaper where they looked at… [Rhonda]: Both papers. [Dom]: …Axona AC-1202? So the finding was in that study, which isrelatively small, that at least not with the diet, but using a ketone supplement that wasformulated with 20 grams of medium-chain triglycerides.So they gave to their patients, I think, justonce a day. And they did show fairly convincingly thatthe elevation of beta-hydroxybutyrate correlated with an improvement in cognitive function,but that correlation was not observed in the ApoE4 positive group, which was a little bit…itwas kind of surprising. I would like to see that study done usingthe ketogenic diet or maybe the ketogenic diet formulated with ketone supplementation. So, the question is…it did not have a negativeeffect, but the question is why didn’t it have a positive effect? [Rhonda]: Was there not another study wherethey actually did the ketone supplement, beta-hydroxybutyrate? I thought there was, but I could be mistakenjust from my lack understanding because that would shed some more light. [Dom]: Yeah, well, they used caprylic triglycerides,C8, which kind of makes a lot of ketones, relatively speaking. [Rhonda]: Unless the ApoE is changing theway you’re making ketones, right? [Dom]: With fatty acids, yeah. [Rhonda]: But they measured, you said theymeasured beta-hydroxybutyrate in everyone and the levels were the same because it’slike I want to understand why there was no positive benefit in ApoE4 carriers becausethat’s the one thing that…it seems so promising, right, that these ketone, potentially ketonesupplements or a ketogenic diet can help modulate Alzheimer’s disease.[Dom]: Yeah, we…and I think you could doit in different ways. My student presented yesterday, she presentedthis week, but she graduated with her Ph.D. yesterday and her work showed that there’sa remarkable increase in blood flow, and previous work has shown that ketone bodies can increasebrain blood flow by 30% to 40%. So that’s another, when you have vasculardementia, when you have a decreased, that being, staying in a state of nutritional ketogenesis… [Rhonda]: Or just brain injury in general. [Dom]: Yeah, it can have a profound effect. [Rhonda]: Did she understand any of the mechanismsor do you know? Can you talk about it? [Dom]: Yeah, we’re looking at that. Well, we did ischemic wounds, which I mean,I guess you could kind of relate to an aged brain, right, with clogged arteries. We did a Doppler blood flow measurement andshowed that it spiked up considerably when we can elevate ketones. One of the things, it was not dependent uponVEGF. So we looked at all the different factors,so VEGF was not increased in the wound.We looked at a couple of different other thingsthat we thought would be increased. Well, the one thing that stood out in thedata was adenosine. So adenosine levels are significantly elevated. Now adenosine is sort of thought to make ussleepy. You know, when we drink coffee, it’s likean adenosine receptor antagonist. [Rhonda]: No way. [Dom]: There’s high levels of ATP, sort of,being used and we think that they may be causing an elevation of adenosine, but we really haveto delve into the metabolomics to understand why that’s happening.Regardless, adenosine is a very powerful vasodilator,and it’s in higher concentrations, significantly, in the blood and that may be increasing theprofusion of tissues, peripheral tissues, and I think that was it. [Rhonda]: That’s great. Did it have any effect on blood-brain barrierat all as you look at that? [Dom]: We haven’t looked at that. My colleague has been, kind of, looking atthat a little bit. We know with fasting and the ketogenic dietthat you can increase permeability to the blood-brain barrier. Things get through faster. [Rhonda]: You mean ketosis through fastingor both, combining the two? [Dom]: Well, yeah, sort of, yeah, fasting-inducedketosis or even the ketogenic diet can help increase the permeability of the blood-brainbarrier to a wide variety of things. So if you are, sort of, the implications fromhis perspective that if you’re getting a chemotherapeutic drug, if you’re getting some kind of drugthat needs to cross the blood-brain barrier that’s impaired in some way, you might beable to get that across faster in a state of fasting ketosis or the ketogenic diet.So maybe if you’re fasting maybe there’s justless stuff in your blood, there’s competition of things getting through and your blood’ssort of clear and you introduce the drug and you get more rapid transport because there’sco-transporters and other things or things that might be in their diet that may be impedingtransport, there’s multiple independent lines of evidence to suggest that being in a stateof ketosis can help better transport things across, but when we talk about permeabilizingthe blood-brain barrier, that’s like a bad thing, right, making it more permeable.In general, though, you get a knockdown ofneural inflammation from the diet and from therapeutic ketosis, which is something theepilepsy world is very interested in. So there’s a PET scan technique that allowsus to look at neural inflammation in the brain and we know that…this is a conference thatI recently came from, that maybe an excellent predictor of when someone’s going to havea seizure. [Rhonda]: Yeah, that would be really coolto do. Possibly see if some of those effects remediatedthrough this pentose phosphate shunt because glutathione in the brain is one of the majorways of producing inflammation, right? [Dom]: Yeah. [Rhonda]: So let’s say you do this in an animalmodel and then you inhibit some of the pentose phosphate enzymes that are using glucose nowinstead to make NADPH and say, “Oh, is that mitigated?” Do you now not have that same effect? It’d be interesting to see if that’s partof the mechanism because that’s…[Dom]: It’s undoubtedly is, yeah. [Rhonda]: Probably, right? [Dom]: I mean, it’s part of that and, sortof, plays into all that. [Rhonda]: So I have another question for you. Thanks for, like, letting me jump all overthe place because I’m super-interested in this and don’t… [Dom]: We need to follow up on the studies. So one of the studies we, sort of, we wantto do, just following back up on that question, is where ketones are being metabolized andwhat their contribution is to normal brain energy metabolism and disease states and hypoxiastates and traumatic brain injury.So that’s a lot to do. [Rhonda]: And those overlap, too, right? [Dom]: That’s the direction of where we’regoing with that research, is understanding that. [Rhonda]: Just to jump back again before Iask you this other question to George Brooks’ thing is that he’s doing some interestingwork working with some physicians at UCLA, I believe, with traumatic brain injury. So these are people that come out with, like,head gunshot wounds. And they are looking at infusing lactate becausehe’s the lactate guy. But ketone bodies will work the same way. [Dom]: I think it would work better from whatwe know. [Rhonda]: Maybe even better. [Dom]: Yeah, I think a cocktail of the twowould be… And I’ve been debating whether or not to reallystudy lactate sort of the same way I’m studying ketones and maybe make lactate esters or lactateand just to see what kind of results we see starting with our oxygen toxicity model andworking from there, which is an excellent model.[Rhonda]: Obviously, with the lactate, youhave to have a intact blood-brain barrier because if no oxygen is getting there, youwill get the lactic acid build-up, right? Because then, I mean, the mitochondria aren’tgoing to be able to use the lactate, which is converted to pyruvate as energy, so itbuilds up, but he has been doing some work and looking at this glucose sparing in thebrain. And in this, of course, ketone bodies wouldwork very similarly, in theory. [Dom]: Do you know how he gets…? Does he use, like, a lactate? There’s alpha L-polylactate, I think. That’s, like, in a sports supplement and someother thing. [Rhonda]: He actually designed it… [Dom]: Oh, really? [Rhonda]: I think he’s the one that, yeah,he’s got a patent on that. [Dom]: Oh, wow. I was using that back in 1991 and -two. [Rhonda]: Cytomax? [Dom]: Cytomax, yeah. [Rhonda]: That’s him, that’s his. [Dom]: When I raced mountain bikes, that was,like, my go-to supplement, alpha-L-polylactate. [Rhonda]: You should get in touch with George,he’s great. [Dom]: I know, wow.[Rhonda]: So he… Let’s see, what was I just going to say? I lost my train of thought. [Dom]: The delivery or the… [Rhonda]: Oh, yeah, so the delivery that he’sdoing…in this specific study, they’re doing it intravenously, so. But I was mentioning the blood-brain barrierhas to be intact, otherwise, it can go the opposite way where it’s actually bad for you. But it would be really interesting to seeso the question is, one, will this lactate or beta-hydroxybutyrate or whatever ketoneget into the MCT shuttle better? So there’s questions of which one gets inthere? And can they be used together? And there’s all sorts of interesting things,but I’m sort of interested in this for my own personal…[Dom]: Yeah, the neuroprotective capacityseems really compelling. I mean, from what we know about lactate approachingit, going to back my old, like, PhD studies or whatever during hypoxia, but it would relateto so many traumatic brain injuries, hypoxia, so you’re mitigating the damage from hypoxia. [Rhonda]: Yeah, and also traumatic brain injuryis inducing damage, and so, glutathione is one of the major ways of mitigating that. There’s been studies showing that, early on,it’s important. If you get that early on, it prevents thewhole cascade so I think that ketones and lactate both seem to have huge potential forglucose sparing, but that, you know… [Dom]: I think chilling the body and infusingketones, lactate with a couple major few co-factors that are part of the bioenergetic use of thesethings would be a home run as far as… [Rhonda]: Yeah, get some students on it. [Dom]: Yeah, these are grants that I’m kindof working on and writing, but I’m not sure the funding agencies are ready to fully…theywant, like, the magic pill thing.[Rhonda]: So to bring it back to the magicpill, I’m kind of interested. So let’s say someone like me that my dietis mostly, I eat a lot of plants, a lot of greens, broad spectrum, carrots, I eat a lotof vegetables. So I get a ton of fiber. I eat a lot of fish. And I do eat, like, beans, so I’m not adverseto legumes or even to oats.I like fiber. Let’s say, I wanted to, I’m, like, super-interestedin beta-hydroxybutyrate because I’ve been following a lot of the research from Eric’slab and I want to get some of those benefits. I want to increase my FOXO3. I want to reduce my lipid peroxidation, Iwant some of that signaling effect. Plus, I want to beta-hydroxybutyrate to bearound to get to the brain, things like that. Can I potentially take a beta-hydroxybutyrate,whatever delivery source powder, pill, whatever it is, and potentially achieve some of thosesame benefits that you get from nutritional ketosis or ketosis from fasting? Let’s say, but I don’t eat a lot of, I don’teat refined carbs, so I’m not getting a huge insulin spike for the most part.So do you know? [Dom]: So, yeah, you want to have your cakeand eat it? But you don’t eat cake, right? [Rhonda]: But I don’t eat cake. No, I just eat fiber. [Dom]: You want to have your carbs. Yeah, that’s that’s really what we’re workingon right now and understanding in a head-to-head comparison to see if we can derive the samekind of benefits from ketone supplementation as we kind of with the ketogenic diet. We know the first kind of convincing studiesof this that we did was with CNS oxygen toxicity in our rat model, and in that case, the ratswere eating a high-carbohydrate standard rodent chow model, and we administered via an oralroute a ketone supplement in the form of a ketone ester. That’s probably one of the more powerful formsof exogenous ketones that we’ve developed. And that had the ability to prevent CNS oxygentoxicity from happening for almost, over 500% delay in that time to CNS oxygen toxicity.And that could not be achieved with fasting,it could not be achieved with anti-seizure drugs. So when it comes to enhancing and preservingbrain energy metabolism in the face of a tremendous oxidative stress that breaks down brain energymetabolism, we’re able to enhance that, preserve that. So we’ve also studied in our animal modelof cancer, metastatic cancer simply giving ketones to the animals on a high-carbohydratediet, it was almost unexpected the level of enhanced survival that we had with ketonesupplementation. [Rhonda]: What kinds of tumor is it? [Dom]: This is a model of metastatic cancerand the primary tumor, it was derived from a glioblastoma, the GBM. And that tumor cell line is so aggressivethat when it’s injected or implanted into the flank of the animal, there’s rapid systemicmetastasis to all the organs and the brain. And the model that was developed by ProfessorTom Seyfried at Boston College.[Rhonda]: Didn’t he publish a paper on…? [Dom]: It made the cover of the “InternationalJournal of Cancer.” Yeah, when I wanted to this study initially,I wanted to do a brain tumor model, like with our diet and our stuff. He sort of wouldn’t let me use the model. He’s like, “Well, use this model of metastaticcancer because it’s the most aggressive thing out there and no one will…” He’s like, “If you create a cure, somethingthat can cure this animal with metastatic cancer, you basically stumbled upon somethingthat has the potential to cure cancer.” So that, kind of, intrigued me. So I knew working with his model would bekind of difficult because the tumor burden…the animal has died so quick from the tumor burden,but it allowed us to screen a variety of things and just using this model understanding thatin aggressive metastatic cancer, the cells are highly glycolytic, and they’re highlyin favor of using glucose and glutamine probably as an energy source.We’re looking at ways to target glutamine,but have not really, kind of, implemented that yet in a combination therapy. But regardless, the animals are eating a high-carbohydratediet with ketone supplementation and it reduced tumor growth and proliferation. We think that the ketones may be alteringglucose metabolism. [Rhonda]: They induce cell death? [Dom]: Well, we think so. There’s more, like, sort of, apoptosis inthe tumors that were there, but just overall, there’s just less tumor growth and less tumorburden and enhanced, most importantly, a 50% to 60% increase in survival time in animalsthat are supplemented with this. And it wasn’t…although the animals tendto eat a little bit less if they’re in ketosis, naturally, which makes a ketone supplementkind of an attractive thing for people that are dieting because when you’re sending energyto the brain in the form of ketones, it has sort of a satiating effect on your body andit, kind of, shuts off that hypoglycemic trigger that makes you want to binge or crave food. [Rhonda]: So is it changing ghrelin and leptinsignaling, or…? [Dom]: We think so.Yeah, we think so. That’s the next thing to look at. It does impact the satiety centers of thebrain. It’s not like you don’t want to eat, but youhave control over your appetite. [Rhonda]: You know what? I just thought of it. Lauric acid, which is C12, suppresses ghrelinin the gut, which is the hunger hormone… That’s kind of interesting, right? [Dom]: Is that specific for lauric acid? [Rhonda]: It’s specific. [Dom]: I knew with certain fats. Really, lauric acid? That’s interesting. [Rhonda]: It is. [Dom]: I’ll look that up. [Rhonda]: Sorry to interrupt. [crosstalk 00:59:39] Let’s continue becausethis is really cool. So you fed them a normal high-carbohydratediet, gave them this ketone ester, as we were talking about. [Dom]: Ketone ester, the same ketone ester,which is. [Rhonda]: The tumor burden was decreased,survival time increased. [Dom]: Yep, yep. And so that was another demonstration, andthe data looked very similar to the ketogenic diet.So we did the study. [Rhonda]: They’re eating less, you said, right? [Dom]: They were eating slightly less, sowe went back. We were thinking, “Well, maybe we’re justgetting a calorie-restriction effect,” because if you have a mouse model or any kind of cancermodel and you overfeed it the tumors are going to grow faster, and if you underfeed it you’regoing to restrict some of the tumor growth. So they were eating a little bit less, buta maybe only 10% drop in body weight. So we went back and we did a calorie-restrictioncontrol experiment, and although there was a decrease in tumor, it was nothing like theketone supplement. So ketones we know undoubtedly they have anti-cancereffects, and it could be maybe through their expression of their gene expression as a histonedeacetylase inhibitor. We think that they inhibit glycolysis. We think that they influence a number of pathwaysassociated with cancer growth. [Rhonda]: Did you measure mitochondrial respiration? [Dom]: Of the tumor and the tissue or…? [Rhonda]: Just, yeah, of, or any tissue, like,so let’s say, did you give them ketones in this…. [Dom]: We looked at ROS production.Yeah, it can sort of knock down reactive oxygenspecies production. [Rhonda]: In the tumor? Or in… [Dom]: In normal tissue, yeah. And now it’s kind of interesting, too, thatin the tumor tissue in previous experiments, I showed that it could knock it down significantly,but I think in the paper that we published, there was a slight decrease in ROS production. [Rhonda]: Yeah, that’s interesting. [Dom]: We don’t know, we do…our experimentsare sort of like a top-down approach. We find out what works and then, we’re mechanisticallygoing after it and we’re doing -omics work metabolomic in particular, and western blotsand assays. So now we’re going after the mechanism, andif we understand the mechanism, we can, kind of, work backwards and tweak the moleculeor adjust the diet in ways that may enhance the therapeutic effects.[Rhonda]: It would be really interesting todo some of… I don’t know if you can radio label theseketone esters and see if, like… [Dom]: Yeah, that’s another thing we’ll lookat. [Rhonda]:…are they being used as a carbonsource for ATP? Are they being used for something else, right,like…? [Dom]: Tracer studies. That’s what we want to do. Yeah, we’re going to partner with a companythat has, sort of, the market on doing these tracer fate associations. I think even doing a 13-Carbon glucose tracerfate association study where we give this, and we give ketones we look at the fate ofglucose in the presence and absence of ketones and see how that may be influencing…[Rhonda]: There you go, is it going to thepentose phosphate? I mean, this is all stuff I really want toknow, so I’m, like, super-excited someone is doing it… [Dom]: And I wanted to do this from the beginning,but I think we want to find out what works first and then now that we’re identified,sort of, things that work with the diet and ketone supplementation, hyperbaric oxygen,I was telling you. We also study, we do a lot of metformin work. We have…I have one excellent Ph.D.Studentand Nate Ward, he’s looking at the effects of metformin and on survival, tumor growthand doing a lot of the cell-based assays. And he’s also looking at dichloroacetate,DCA. So it activates pyruvate dehydrogenase. So he’s looking at each one, individual, andalso in combination as a cancer therapy. [Rhonda]: Okay, I’ll give you some of my ideas. And so I, just because I want someone to testthis. But with DCA, so I did a lot of work, in mygraduate research, I did a lot of cancer metabolism I was in contact with Craig Thompson, RalphDeBerardinis … all those people that we were talking about earlier, and they did alot of glucose withdrawal studies, glutamine withdrawal studies, blah, blah, blah, blah,like all that stuff.So I’ve got a lot of interest in it stemmingback from years and years ago. And I also was very active in apoptosis, workingwith some of the top guys in the field, Doug Green. So I, sort of, like, the way I think aboutall of this and the intersection between them is that, like cancer cells are… Here’s why I think cancer cells are glycolytic. So I mean, the Warburg effect you’ve talkedabout this and you published on it.[Dom]: Is it a cause or a consequence? Yeah, damage respiration. That’s sort of a… [Rhonda]: Right, so I don’t think…and Ithink even Otto Warburg himself published immediately after his original science paperbecause he originally theorized damaged mitochondria were the cause it, but then it’s not. It’s not that the mitochondria are damagedenough that they’re not respiring.Even to this day, I don’t think we have reallyshown that or disproven that, like, thoroughly. I don’t think it’s a cause, but the reasonI think that cancer cells become glycolytic, I don’t know what causes it or how, I thinkthe reason is they do is because they don’t want to use their mitochondria. And the reason they don’t want to use theirmitochondria is because mitochondria produce ROS, and that will drive… This is the whole basis of how you kill acancer cell, chemotherapeutic drugs, the way they work is because they induce a littlebit of reactive oxygen species toxicity through a variety of mechanisms. [Dom]: Some more than others, but, yeah, [Rhonda]: Right, through a variety of mechanisms… [Dom]: Augment oxidative stress, yeah. [Rhonda]: …which then pushes the cancercell to death because normal cells don’t have boatloads of proapoptotic… If you look at any cancer cell, they have,like, boatloads of it.I mean, just tons and tons of proapoptotics,but they have countered it and they are ready to die. They just need a little push, and DCA activatesmitochondria, and I think that’s part of how… [Dom]: Hyperbaric oxygen, too. [Rhonda]: Hyperbaric oxygen, and we were talkingabout this earlier. I think that’s also… [Dom]: Naturally stimulate it, yeah. [Rhonda]: Yeah. But so I’d like to see someone, sort of, testthat because I think that possibly DCA wouldn’t be as potent at killing cancer cells if yougave it NAC or something that’s going to sequester the reactive oxygen species. [Dom]: Yeah, so could you block it, yeah. That’s another interesting, that would bea good control to do, yeah. [Rhonda]: And studies have shown that givingmice supplemental vitamin E, something that’s going to potently sequester reactive oxygenspecies, actually allows tumors to grow faster. And this has been [inaudible 01:06:35] inresearch.[Dom]: Yeah, N-acetylcysteine, too, and NAC. [Rhonda]: NAC, as well, yeah. [Dom]: With metastatic melanoma, I think,came out, yeah. [Rhonda]: Yep, yep. And then also there’s one in lung cancer. I think it was the same group publishing that,too. But so I think that part of the reason…becausecancer cells are so smart that I think that not having their mitochondria active is verybeneficial to them because. [Dom]: Or less mitochondria, too, as… [Rhonda]: Yeah, less mitochondria. [Dom]: So just a deficiency. And there’s, yes, reports. So there’s debate are the mitochondria defectiveor are there just a decreasing number of mitochondria? I think it’s both. I think mitochondria are structurally andfunctionally impaired, and I think there’s a deficiency of them. [Rhonda]: You mean in cancer cells or [crosstalk01:07:21] normal? [Dom]: I think in cancer cells, but I thinkin just normal cells, if you if you’re drinking alcohol and bombarding the liver with oxidativestress that you’re damaging mitochondrial…you’re damaging the mitochondria, and over time,you are going to…[Rhonda]: Of course. Normal metabolism. [Dom]: …induce the Warburg effect by causingprogressive damage, mitochondrial oxidative phosphorylation the cell’s ability to maintainenergy status through mitochondrial oxidative phosphorylation will be, its capacity willbe impaired. So it will activate oncogenes and oncogenesthat drive the glycolate. You’ll have, some cells will die, the onesthat survive are the ones that go on and activate the complementive genes that cause the transformationof a normal cell to a cancer cell. And that’s how I, sort of, say that this progressivedamage, you could do it with radiation, you could do it with chemotherapeutic agents,you could do it with inflammation, chronic inflammation is damaging respiration. And it’s that damaged respiration that cankill off cells and the ones that survive, that are hardy enough to activate the geneticprogram that allow them to survive are kicking on the oncogenes that will then go on andproduce a Warburg phenotype.So that’s sort of the metabolic theory ofcancer in a nutshell. And it differs from what the thought leadersin the field are saying that it’s more of default state to ensure the preservation ofthe cancer that that exists, but not the cause, but there’s still this elusive enabling factorthat we still don’t know and I think the metabolic theory nicely explains is a pretty elegantexplanation as to how a normal cell converts to a cancer cell. And there are other genes involved, definitely,but, I mean, at the very most, the most we can link hereditary effect to cancer is maybeabout 10% 7%, I think, was a number that is being thrown around now, but about 10% ofcancers are from hereditary, but this, the epigenetics, I think, is something that will,yeah…and that’s something that’s evolving very fast.[Rhonda]: I mean, I think that DNA damage,and that’s been pretty well-shown, damaging DNA, both mitochondrial and nuclear DNA, thedamage that can lead to aberrant cells metab-, like, cells. [Dom]: And I think the mitochondria are moreimportant because the mitochondria have less of a robust DNA repair mechanism. And also the DNA of the mitochondria havemore coding regions. So if you bombard cells with radiation classically,radiation biologists are taught that that radiation is directly damaging nuclear DNAand then that kicks on, causes the genomic instability that causes cancers, but I thinkwhat is being more appreciated now is that the mitochondria are selectively vulnerablebecause their DNA repair mechanisms are far less robust. They have much greater coding regions withintheir DNA, and they are the ones kind of calling the shots, they’re making the energy, andif the energy status of the cell goes down, that’s going to trigger the nucleus, that’sgoing to trigger an energetic crisis in the nucleus, and the nucleus is going to kickon oncogenes to transform the cell from a normal to a cancer cell.So the stability of a nuclear genome is tightlyregulated to the energetic state of the cell. [Rhonda]: Yeah, so I have a little bit ofa different way of thinking about it mostly because I’m also doing a lot of research onthis experimentally. So I measure damaged DNA, and I measure mitochondrialfunction after I induce radiation in some sort of… [Dom]: In primary cells? [Rhonda]: In humans, in blood cells, yeah. But so, mitochondria, you mentioned that nuclear,they have more repair mechanisms, and that’s true, but mitochondria have very elegant andbeautiful way of repairing damage through fusion, right, mitochondrial fusion and fission. And this is a process, I mean, this is howwe are able to repair damaged mitochondria because they’re constantly fusing with healthymitochondria changing, I mean, exchanging their DNA content, protein, things like that,and fissing back part.So, of course, when those mechanisms becomeimpaired, then that’s, we start to have more accumulation of damage more because they can’trepair. [Dom]: Fission proteins, the production ofthe proteins that cause that are also tightly linked to oxidative stress. [Rhonda]: Yeah, so I mean there’s lots ofdifferent ways to repair damaged mitochondria. I also did a lot of work in graduate school. But I don’t think it’s clear, I don’t thinkthat the metabolic theory of cancer… [Dom]: Far from clear. [Rhonda]: You know, when you drop the ATPstatus in the cell, what happens is the cell dies and apoptosis gets trigger, and that’sthe primary…before oncogenes are activated, the cell dies. [Dom]: Unless it’s if it’s more gradual, thenyou have the activation. And most cells die. So you have 999 cells die and then you haveone that, kind of, activates the complement of genes that can, allows it to survive, givesit survival advantage.That’s what you get with chemo, too, or radiationwhen you blast a tumor with radiation. You get, there’s few that can survive. And if you do that over and over to the tumoror over the course of chemos, you’re kind of making a super-cancer because you’re increasinglyselecting for the most aggressive, glycolytic hardy stem cell-like tumor cell by hittingit with more chemo, you’re just causing more DNA damage and more transformation, mutagenicity. Do you see it like that? [Rhonda]: I don’t know. [Dom]: I’m not against standard of care, butI’m in favor of. [Rhonda]: I wanted to believe, in graduateschool, I wanted nothing more than to believe that mitochondrial dysfunction is the causeof cancer, but I just couldn’t, just couldn’t attribute to myself. You know, I kept trying and trying, it wouldhave been easier for my graduate…My graduate career would have been shorter,for one, but I just couldn’t enough evidence to convince myself of it. And that doesn’t mean that it doesn’t, it’snot true, it just means, I just…so far don’t…I don’t think that’s the origin of cancer. I think that metabolic dysfunction plays avery important role in causing cancer.Most primarily through inflammation throughall the effects of, like the insulin signaling and the inflammation, the reactive oxygenspecies. All these things that are damaging the cell,but I don’t necessarily see it the way that you, sort of, described it as them… [Dom]: An initiator. [Rhonda]: Yeah, them changing or activatingoncogenes. I don’t think that’s really been shown. [Dom]: I don’t think anyone’s studying thatbecause…or studying it in the way that would make it clear, and I think it may vary betweencancers like leukemia and lymphoma and relative to glioblastoma. I mean, we know these are just, they havea different metabolic and a different gene signature. Glioblastoma has hundreds, if not thousands,of genetic mutations. You know, so hence the name glioblastoma multiforming,you have all these different cell types and everything. Whereas other types of, like, for leukemia,for example, Gleevec works marvelously well because it’s targeting something that’s veryspecific.So I think it will be impossible to get aclear answer to this and I don’t think it’s… I think maybe I’m a centrist. So I’m somewhere in between the genetic versusmetabolic, but leaning more towards a metabolic origin for many solid tumors, but there’ssome cancer that just, kind of, throw me a curveball like different types of lymphomaand leukemia, testicular cancer. And they’re all responsive to chemo, manyof them are. [Rhonda]: So what about, like for me, whenI think of mitochondrial dysfunction, to me, that leads more to neurological dysfunction,neurological disorders, less for cancer, less of cancer like when you…mitochondrial mutations,genetic mitochondrial mutations in mitochondria genes, there’s much more it has much moreof an effect on causing certain types of neurological disorders rather than, like, cancer, right? There’s one, I think, the succinate dehydrogenasecomplex II, one of the components.I know this because I was trying to figurethis out in graduate school, it was, like, a huge question. [Dom]: And the really bad diseases, yeah,these mitochondrial disease, yeah. [Rhonda]: So that’s what I usually think of,when I think of mitochondrial dysfunction, I always think of it as being more of a, playinga causal or initiating role in neurological disorders and neurological dysfunction, andnot so much as, plays a role. I think that mitochondrial dysfunction andabnormal metabolism plays a role in cancer, but I don’t think it’s that initiator theway you were describing it.I just don’t think that’s been shown. [Dom]: I’m going to prove you wrong. Well, our lab is we’re not, we’re just tryingto find the answer. And I think that as we move forward and developthe tools, I think the answer will start to get a little bit more clear at least usingthe models that we’re using. But I think regardless even of the origin…theorigin is important because if we…the way we treat and prevent cancer is going to bedifferent if we know the origin for sure. You know, if it is a mitochondrial versusgenetic origin or whatever origin.And, I mean, there’s a case for viral originsof cancer, too, but these viruses are, sort of, the ones that damaged mitochondria, too. I’ve been, sort of, interested in the viralorigins of cancer. It will influence how we prevent cancer. So in addition to developing therapies, wewant to study animal models, and maybe inducers of carcinogenesis and then adapt them, orto treat them prior to, or put them on a therapy prior to letting them live out their livesif they’re prone to spontaneously forming tumors, or letting them live a few monthsprior to the introduction of a carcinogenic agent, and then seeing after X amount of time,whether the tumors form.So can we prevent this does have profoundimplications for people who have been, went through chemotherapy or had cancer in thepast, and should they be on some, kind of, preventative, should they do a therapeuticfast? I get this question a lot for four or fivedays to a week every two or three times a year. Should they do that? Will it help them purge their body of pre-cancerouscells and put that metabolic stress? And these are important questions that noone is really trying to answer at least from a point of a prevention, sort of, idea.So I think that’s sort of on my horizon asthe next big thing. Can we develop and implement have these protocolsavailable for someone to do? It could be intermittent fasting. I personally like ketogenic intermittent fasting,where you’re taking in ketogenic fasting, ketone supplements throughout the day andthrough 20 hours of the day and you have a 4-hour window where you eat a well-balancedketogenic meal that’s rich in vegetables and high in fats and protein. And I think that could be something that couldbe implemented and something that I personally am interested in writing up a protocol forthat. The work, the studies done with metforminand showing that people who, type 2 diabetics that are taking metformin have a 62% lesschance of getting pancreatic cancer. We need to study that, you know? Should that be a part of a comprehensive preventativetherapy that people should do? I mean, I question, should I have my parentson this? Like, should they, you know…? [Rhonda]: On metformin? [Dom]: Or yeah, on metformin, or my mom actuallyhad cancer years ago.Should she be on something like this? Should everybody be after the age of 50 ifmost of their family members have died of cancer? [Rhonda]: Have there been any long-term studieson the effects of metformin? Because I’m very interested in it, but I’malways hesitant with any drug or anything that’s perturbing biological systems. [Dom]: Yeah, well, there’s hundreds of thousands,if not millions, of people on metformin so I’d say, yeah, there’s a long-term data outthere whether…and a lot of retrospective studies have been done. It’s a relatively safe drug. Lactic acidosis could be a problem in higherdoses for some people maybe with renal insufficiency or impaired liver function. And then, another thing that creeps up couldbe vitamin B12 deficiency. So if you are…our ability to absorb vitaminB12 as we age is decreased, so maybe a sublingual form or even B12 injections in people thatare older? [Rhonda]: Why does metformin affect B12? [Dom]: That’s a question I don’t know an answerto, but I can speculate that it may influence the transporter, and it also tends to makestools loose for some people.Things go through you a little bit fasterand impairs… “Impair” is not a good word. It changes the gut microbiome favorably. So “Nature”….the paper there’s that cameout about two weeks ago showing that there’s a favorable shift in the gut microbiome… [Rhonda]: Now the reason why it’s interesting. [Dom]: …with metformin, and that may explainit’s type 2 diabetic it’s glucose-lowering effects. That sort of hit me as, “Wow, I had not reallygiven that a whole lot of thought, but it’s something that I think we should be lookinginto.” So I was like, “Yeah, we need to collect allthe start collecting the feces from these animals that we’re doing metformin on to figureout what’s going on with the gut microbiome,” but I think it’s influencing the absorptionof B12 in some way that I don’t really know. [Rhonda]: Does metformin…is it doing anythingin addition to mimicking a lot of the same signaling pathways that caloric restrictiondoes? Like, is there something additional that…youknow? [Dom]: Yeah, yeah, AMP kinase, for sure.So without a doubt, I mean, it’s mimickingmany of the pathways of associated with calorie restriction and with fasting. To what degree it’s mimicking that relativeto a length or duration of fasting? I don’t know. We’re doing some work right now looking atAMP kinase and mTOR, and downstream and upstream signaling insulin and these things, and tryingto get a picture of this, at least in a rodent model. And then, I’d like to ultimately replicatesome of this stuff in humans, but what I think, I think metformin would be best used maybein pulsing it a few times a year. A lot of these things, metabolic interventionstend to work best when you cycle them, I think. And I really have not been doing that, butI think it’s a theory that I have been working on.I need my scrapbook. [Rhonda]: Why do you think that is? [Dom]: Because your body is similar with ketoadaptationthat your body can, kind of, reset to that level, initially fasting on the ketogenicdiet is sort of a stress and it can induce a hormetic effect in gene transcription andthen we, sort of, get used to that. You know, our gluconeogenesis is upregulatedto that level, but I think it’s good to maybe pick probably not a high-carb diet, but maybea Paleo diet a low-carb ketogenic diet and maybe something in between and do some intermittentfasting on occasional days. So I think this promotes metabolic flexibility. It allows our body to adapt to different situationswithout being, kind of, overwhelmed by the stressor of it. So I think, to some extent, it is hormesis. And interestingly, metformin causes mitochondrialstress, and actually, mitochondrial damage. Some researchers coined the term that it’sstimulating reactive oxygen species production and causing mitochondria dysfunction, metforminis, and this is kind of well known in the field. So the general feeling is that, “Well, ifI take metformin and I go exercise, why is it not killing my exercise capacity or myVO2 max or making me lethargic or tired?” It’s not doing that, actually I think it’senhancing.There was a [crosstalk 01:25:34] study. [Rhonda]: Does it affect biogenesis? [Dom]: It does. So yeah, so the thought that it’s kind ofstimulating, there’s a hormetic effect. It’s damaging the mitochondria, some peoplebelieve this, and you get a secondary, yeah, effect through that way, like it’s, kind oflike an exercise drug. But I approached it from the perspective thatmetformin could lower blood glucose at least if it was high and it activated AMP kinase,and it may decrease circulating insulin. So I approached it as a cancer drug from thatperspective, but the more conferences we go to, there’s a plethora of data coming outof metformin and a lot of people are studying it from the perspective of impaired complexI or complex II activity in the mitochondria. So they’re looking at it from that perspective. [Rhonda]: Interesting.[Dom]: I know. [Rhonda]: Yeah, it’s super interesting. Especially, you’re giving its effects on longevityand cancer. [Dom]: Yeah, yeah, and I think our most recentdata did show an increase in ROS production in our cell line. [Rhonda]: I wonder if that’s how it’s alsokilling the cancer cells. [Dom]: Yeah, it could be. [Rhonda]: You mentioned when you were talkingabout gluconeogenesis, you triggered something in my mind. I wanted to ask you, I forgot. So when you’re in nutritional ketosis or fasting-inducedketosis, you need to make glucose you still need glucose, your red blood cells have nomitochondria and your red blood cells are important, right? So you’re making glucose through this processthat you mentioned called gluconeogenesis.[Dom]: Glucose does not bottom out. It’s not like one or the other. You’re pulling fuel source from… [Rhonda]: So I wanted to ask you about, like,how… Has anyone looked at where…so if you areon a pretty strict ketogenic diet or whatever it is you’re doing to get into ketosis, what…sodoes the liver use, like, glycerol, lactate, like, both as a primary source to make glucose? Is that glucose predominantly going to redblood cells or does it go has that been looked at to see, like, where, you know…? So red blood cells, like, are they gettingenough of their glucose or they, you know? [Dom]: I think so.I mean, you’d probably have to severely calorierestrict. In those cases, you could become anemic orimpair…your immune system is also, too, highly dependent to some extent on glucoseand glutamate. So, yeah, you have lactate, you have the glycerolbackbone, the fats… [Rhonda]: But they always have mitochondria. [Dom]: Yes, yes. So glycerol backbone of fatty acids or oftriglycerides, for sure, lactate, yes, and amino acids, gluconeogenic amino acids inyour diet also are a source of glucose. So gluconeogenic amino acids in your skeletalmuscle your muscles constantly breaking down or remodeling especially in athletes.So they’re all sources. The contribution of each of these gluconeogenicsources in each individual probably varies tremendously, but I would say that… So glucose is always going to be there, andthe body ensures through very powerful homeostatic mechanisms that your glucose is going to stay,rarely go below three, maybe 2.5 millimolar, mine will drop two, go to four and stay withina very tight range, but what does change considerably from a glucose regulation standpoint is theinsulin. Insulin bottoms out to the point where I’veseen enough blood work to show that in many cases, insulin and IGF-1 is below the referencerange.So insulin signaling goes down. So if insulin’s down, all those insulin pathwaysthat you see on your flowcharts are all going to be suppressed and IGF-1, obviously, it’sgoing to be lower and I think that’s a really important consideration to factor in as itrelates to cancer therapeutics, cancer biology, cancer prevention, even. But also from the perspective of muscle metabolism. And I think by keeping insulin signaling sortof low, you upregulate factors that make you more responsive to insulin. So I think, and ketones can kind of compensatefor a deficiency in insulin, and that was, a lot of the reviews by Richard Veech talkedabout that.And the ketones themselves are anti-catabolicfor protein sparing. So if you’re in a state of ketosis, you’reprotecting gluconeogenic amino acids and skeletal muscle from being degraded. So you are as a metabolic fuel, but you’realso, there’s evidence that you’re inhibiting proteolytic enzymes and pathways that wouldotherwise be chewing up your muscle tissue over time. [Rhonda]: That’s super-cool. [Dom]: So it’s anti-catabolic, yeah, so ketonesare anti-catabolic in that part. [Rhonda]: So then you’re probably not using,I mean, the gluconeogenic amino acids as much… [Dom]: From skeletal muscle, yeah, not asmuch.So the idea is that you want to keep pumpingin the fat, too, if you’re on a ketogenic diet. If it’s not sufficient with ample amountsof fat, you’re probably much more catabolic. So you want to ensure that you’re using thefatty acids, go to the mitochondria that uses fuel, they keep the mitochondria happy asdo the ketones. Then the glycerol is kind of,shuttled andit’s a very nice kind of an elegant pathway to ensure that we have that flux of glucosefor vital functions like the red blood cells and making…there’s an number of neurotransmittersand hormones that require a baseline level of insulin or glucose to be used.[Rhonda]: Yeah, and we talked about… [Dom]: A lot of things, yeah. [Rhonda]: Yeah, it’s just so many things todiscuss, but I’m really, like, thankful that you… [Dom]: We can probably talk for, like, fouror five days non-stop before we like collapse from… [Rhonda]: You see how I get, I get, like,really excited and I’m like, “Okay, wait, I got to ask you this question. I have this idea.” And then you’re just, like, full of information.So it’s kind of neat. [Dom]: A fun field to be in, right? [Rhonda]: Totally, I guess. [Dom]: I mean, I’m always, I feel like I’mso lucky to be in an area of…be in an occupation where discovery, we have the potential todiscover something new that can impact the population and get paid for it. [Rhonda]: Okay, that part, I get. Because you totally, I’m sorry to change thesubject, but you’re talking about glutaminolysis and I have done a lot of research on this,and there are questions that I would love to be answered, but haven’t been. So, since you’re looking at this and you haveresources, I’ll just throw it out there. Obviously, you said this is well-known literaturethat glucose and glutamine are both source…cancer cells love them. It’s like crack for cancer cells, both glucoseand glutamine, and I’ve done a lot of studies on various types of cancer cells and theseare in vitro.So this is not in an animal model where Ican, I withdraw glucose and the cancer cells will proliferate slower, some will die, butif there’s glutamine there… [Dom]: What’s your level of glutamine? [Rhonda]: Two millimolar. [Dom]: Two millimolar, yeah. [Rhonda]: Yeah, so then I would start withdrawingthe glutamine and glutamine withdrawal, this is all in vitro, though. Glutamine withdrawal would kill them within24 hours, but. [Dom]: Pretty lethal, yeah. [Rhonda]: Very lethal, and has been shownat least some of the studies that were initially done by Ralph DeBarardinis when he was withCraig Thompson, and later when he established his own lab where he radiolabeled and showedthat, actually, it was being used predominantly for macromolecular synthesis and not for whichis…of course, that makes sense because a lot of tumor cells aren’t using the mitochondria.[Dom]: Making fatty acids, actually. [Rhonda]: Making fatty acids, proteins, like,for new synthesis. [Dom]: Like, cell membranes and stuff. [Rhonda]: Right, so the question for you…thequestion that I have and this is… So that’s one of the spectrum. Okay, glutamine seems bad when you’re lookingfrom an in vitro perspective and I did these studies, but many people have published onthis. You’re familiar with the literature. But then there’s the other perspective wherereally glutamine really is believed, like, to gut, to cells, it’s very, very healingand therapeutic for gut, and when you take glutamine orally, the gut takes it, it’s notgetting into your bloodstream, It’s not being so…[Dom]: The gut and the liver take its shareand very little of it actually gets into the bloodstream. [Rhonda]: Right, so that’s what I’m gettingat. The question is, if you have a mouse modelof a solid tumor that’s not gut-oriented, so it’s not colon cancer, like let’s say it’syou got a pancreatic cancer or… [Dom]: Brain tumor. [Rhonda]: …brain tumor, then you give themouse glutamine, is that really is it really going to affect the tumor or is it just goingto help the gut? I mean, of course, it can indirectly affectit, but the question for me, in my mind, is, well, yeah, if you had tumor in the gut, man,that’s like crack for the tumor.Do not take glutamine, do not, you know… But on the other hand, if you’ve got gut issues,you know… [Dom]: It can be helpful. [Rhonda]: Right, do you see what I’m gettingat? [Dom]: This is something that I have thoughtabout. [Rhonda]: You have? [Dom]: Yeah, I think about stuff like this,yeah. [Rhonda]: I’m not alone, yeah, well, in vitrois very different because the way our bodies are working and the way glutamine when wetake glutamine, it’s affecting our gut, it’s very important. I mean, it helped. It’s helped me. [Dom]: I used to take it. [Rhonda]: It’s helped me with gut, but thenthere’s this whole, like, conflict in my head around…cancer cells love it, but the questionis if I’m taking it orally, and I have some cancer cells, in my, I don’t know, my liveror something, then I guess you said liver is one that does.It does use it, but, so the question is, isthat harming me or is it helping me? [Dom]: Should you take it or not? Yeah, I get that question a lot. For GI cancers and liver cancer, I would saydo not supplement glutamine, and I would say under most conditions…I always say in thosestates, I actually tried to look up the glutamine content of food, and you might want to avoidit or minimize glutamine, high glutamine-containing foods. Otherwise, I wouldn’t really pay too muchattention.Some patients really stress out about it butI think if you just keep your protein low to moderate, or keep your protein at a levelto ensure proper regeneration and just, kind of, replenishment of your normal cells, andprevent protein deficiency, and being in a state of ketosis will help with that to someextent, but glutamine is pretty low on… [Rhonda]: The classical ketosis, right, whereit’s 10% protein? [Dom]: Yeah, yeah, and I think that will loweryour blood glutamine levels, just being on a ketogenic diet will do that. And then, you could further lower it by selectingprotein food sources that are lower on the end of, are glutamine.I’m not for avoiding protein types of supplements,avoiding glutamine supplementation all together. And you may be able to further suppress glutamineby taking a supplement that’s like high branched-chain amino acids, high essential amino acids. So taking a supplement that is formulatedin a way that, kind of, gives you essential amino acids, excluding glutamate, of course. Glutamine is not an essential amino acid. It’s conditionally essential. But then you…I don’t think you’ll run therisk of being deficient in glutamine in any way, but I would avoid, I would pay attentionto it if you have a GI cancer or liver cancer, and then if… [Rhonda]: The liver, I didn’t know, but yeah,GI was… [Dom]: So if you have, say, for example, like,a brain tumor and you’re taking a drug that can impair systemically, you’re taking somethingthat impairs your GI function, and it may be helpful to take a little bit of glutaminebecause I don’t…I really don’t think…the gut’s going to be very greedy when it comesto glutamine.[Rhonda]: It’s very greedy. [Dom]: So I think just maybe even 5, 10 gramsof glutamine to help repair your gut. We know that if your gut permeability is impaired,that can wreak havoc in your body as far as systemic inflammation. So try to…and there’s other ways to repairyour gut, too, but I think glutamine may be a factor in helping to ensure proper gut.[Rhonda]: Yeah, there’s definitely other ways. I mean, I think that you know, like I wassaying, fiber, good diet, and things like that. [Dom]: Exactly. But glutamine has been used in oncology. So, yeah, glutamine is for helping peoplewith chemo combating the issues with chemo. And then glutamine has almost been like astaple, you know… [Rhonda]: So they give it to chemo patients,kind of… [Dom]: Help them recover part of the immunesystem, too. [Rhonda]: Because your gut regulates the Immunesystems well. [Dom]: Your gut is, like, what, 70%, 80% ofyour immune system, right? So it’s huge. So we want to keep your gut as healthy aspossible. And there are many drugs out there that reallyimpair gut mobil-, or… [Rhonda]: And diets, too. [Dom]: Yeah, and diets, too. [Rhonda]: Yeah, so, cool. All right, Dom. [Dom]: Endless amount of things we can talkabout, right? [Rhonda]: I know, right. Just keep going, Dom. [Dom]: Yeah, I’ve had, like, 100 things popinto my head.I was like, “Should I bring that idea or shouldI not? No, no, no.” [Rhonda]: Bring it up, I’m always interestedin new things, so… [Dom]: We do have ketone esters here and nottoo many people are brave enough to try them, but we have a lot of studies going on usinga wide range. See, they’re all different types of ketonesupplementation, and I know that’s, kind of, was like one of your main interests that youwanted to hit on. [Rhonda]: Yeah, yes. [Dom]: And I would say stay tuned becausewe have so much going on right now with, like, all these studies looking at ketone supplementationand the answer that I want to really focus on is what, kind of, benefits are we deriving? There’s been so much work on the diet whatkind of, benefits can we mimic with just purely a ketone supplementation? And can we further augment the therapeuticefficacy of the ketogenic diet with supplementation? So with diet and then we supplement 10%, maybe20% of the calories with some form of ketone supplementation.Hopefully. we work to formulate it in a way that makesit pleasurable to taste and not taste like gasoline, if we can do that. [Rhonda]: Does it taste really like gasoline? [Dom]: Yes, yeah, like jet fuel, really. It’s pretty bad, but the ketone esters do,but we’re working on the ketone salts that you might know of, KetoCaNa, ketone, there’sa couple ketone products out there. [Rhonda]: Are there benefits or drawbackstaking one or the other? [Dom]: The salts are like as far as ketogenicpotential, are pretty similar to MCT.So they’re another level up from MCTs, I wouldsay right now, but they’re being formulated. We’re formulating them and testing them inways to make them closer in potency to the ketone esters. And I think… [Rhonda]: Are these available, like, to consumers? [Dom]: Not for human use yet, but we’re workingon doing all the safety studies and then all the palatability work and formulating themin a way that probably within the next six months to a year, that they will be out.[Rhonda]: Oh, so soon? [Dom]: We’re tracking them for therapeuticpurposes. So for clinical trials, for specific diseases,and then, they kind of work backwards to the consumer broad market eventually. But, and for oxygen toxicity, obviously. That was the original application for oxygen… [Rhonda]: Yeah, I’m also really interestedin it just for movement disorders. My mother has orthostatic tremor, and she’snot the kind of person that is very compliant. I mean, she may try something for, like, acouple of days, maybe a week. [Dom]: That’s typical. [Rhonda]: Very typical, and drives me crazybecause I feel like I have so much knowledge, I could help her, and it’s just very hardto get her to do, to get any movement, but I would…I’m very interested in the potentialuse of ketone esters or whatever delivery method that’s the best in potentially helpingreduce her tremor because when she stands still, her legs shake and it’s very, it’sinhibiting to her life, I mean having to…When she’s walking, I mean, she’s fine, butif she stands, stands in line… [Dom]: From sitting to standing, and just… [Rhonda]: No, even walking to standing, juststanding. So standing still, it’s called orthostatictremor, and it’s common enough… [Dom]: Yeah, yeah, I’ve heard about it. [Rhonda]: …that, but it’s not like, it’snot as common as essential tremor, but she also has essential tremor, as well, [crosstalk01:43:13] kind of interesting. Yeah, so I’m extremely interested in the potentialbenefits of nutritional ketosis, yes, but, like I said, she’s not very compliant. So I’m, sort of, like, okay possibly givingher some sort of ketone ester see how that would affect…because then again, if she… [Dom]: You might want to start with MCTs. I mean, something, what I showed you today,the MCT Powder by Quest Nutrition is really pretty close it’s very potent from a ketogenicperspective and if you were to take four to six to up to eight scoops a day, which wouldbe tolerable in a course of a day, she would be in a mild state of ketosis and would begetting the benefits from it.[Rhonda]: Really? Do you think that would be easier than, like,the MCT oils? [Dom]: From perspective of GI tolerance, yeah. Many people I would say up to 40% or 50% ofpeople are going to have some tolerability issues with liquid MCTs. At least a big dose that gets you up intosustainable ketosis. You can incorporate MCTs in your food, evensalad dressing is cooked with it, mix it in with different things, but the MCT powderI found was, is you can get levels about twice as higher than you can with the oil just simplybecause your GI tolerance is much better in a powder form.So it’s formulated in a way that, kind of,allows us to sustain the slower release of the MCT instead of a liquid which tends to…somepeople just can’t tolerate the liquid at all. I can tolerate fairly high amounts relativelyspeaking, but I could tolerate much higher amounts with an MCT powder. [Rhonda]: You can put it, like, and you canmix it with water, coffee, tea or whatever. [Dom]: Yeah, I could put it in coffee. [Rhonda]: Yeah, [inaudible 01:45:05].. [Dom]: Yeah, and you liked it, right? [Rhonda]: Yeah. [Dom]: I mean, it’s like great. [Rhonda]: It’s like creamer, but, you know. [Dom]: So they really nailed that product. [Rhonda]: The coffee makes her tremors worseso she doesn’t drink caffeine. [Dom]: No stimulants, yeah. What about decaf coffee or something likethat? [Rhonda]: Um, I think she… [Dom]: It’s a good vehicle for MCTs. I mean, you could put, like, butter and MCT. I know you don’t like sweeteners, but I justput in a little pinch of stevia in there.[Rhonda]: Stevia is okay. [Dom]: And it makes it a really enjoyabledrink for me. [Rhonda]: Yeah, that’s great that it’s availablebecause I’m, kind of…that would be something that we probably try, seeing if that has anyeffect on her tremors. [Dom]: As you know, diet is key, though. [Rhonda]: Diet is key, yeah, because if you’re,like, eating a bunch of refined crap and processed foods, and just terrible diet, it’s not muchthat ketones are going to do. Right? [Dom]: Yeah, and what I find in people thatare resistant is that if you can introduce foods that replace other foods which is good,and I think Quest Nutrition, too, is making a line of…They’re not out yet, but I’vetried everything from a ketogenic Oreo to a ketogenic brownie to ketogenic chocolatebars. So these are foods that when you eat them,you’re in ketosis, and they taste as good or good as their high-sugar counterparts thatare on the market. So I see that as almost like the next frontier,like, designing, developing ketogenic food products from whole food ingredients thatare from natural sources and not synthetic sweeteners or artificial flavors and thingslike that, that will allow you to…will ensure greater compliance of nutritional ketosis. Mainly at first, maybe targeting disease populations,but undoubtedly people from all walks of life will be wanting to use these foods especiallyif they taste good. And I can tell you from kind of a beta testerpoint of view that it is possible to create a line of food products from crackers to chipsto, you name it, really. It’s possible to…it’s pretty easy to makesomething taste good when you’re working with fat because fat has…fat and salt kind ofmake things, are really good on our palate.And they’re very satiating. So we’ll eat it a little bit, and it’s justenough to sustain us and give us the energy that we need without overeating. [Rhonda]: Yeah. [Dom]: So I’m excited about the ketone supplements,obviously, but I’m excited about a line of ketogenic diet food products that can ensurecompliance in people who really need it. Because I say that’s where…there are peoplewho know the diet would help them from a therapeutic standpoint, but they just lose interest aftertrying to follow through with the diet.[Rhonda]: Yeah, and it would help eliminatea lot of suffering especially for some of these people with these, like you said, drug-resistantseizures. [Dom]: Yeah, for sure. [Rhonda]: That, so, yeah. Well, super-cool, Dom. Thanks for speaking with me and for doingall this really cool research. I’m going to keep following your researching. [Dom]: Thanks for visiting. [Rhonda]: If you want to learn more aboutyour research, what you do, things you talk about anything related to your research, wherecan we hear more about you? [Dom]: I would say I’m working on a more interactive,but broader site, but for right now, ketonutrition.org, I think, would be the site to go where I compilea bunch of links on there with dietitians, ketogenic-savvy, registered dietitians thatI recommend, books, talks from IHMC, which I’m excited to listen to your IHMC talk thisweek because that’s on there.So I would say that ketonutrition.org wouldbe the site… [Rhonda]: Ketonutrition.org. And what about social media, do you have any? [Dom]: Yeah, Facebook. You can find me on Facebook, on Twitter, LinkedIn,Pinterest, maybe I go there sometimes. But Facebook, Twitter, and LinkedIn are siteswhere I will post information about our research or related research in the area of nutritionalketosis and metabolism. [Rhonda]: Awesome. Thanks a lot, Dom. [Dom]: Thank you. Thanks for having me..

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