So, today we are going to have the best presentations of all of this endocrine society meeting because it's going to be about vitamin D. So, I would like that you take a look of the QR code. Could you please put back the QR code? So, if you have any questions and online questions, so we can see it over here and read it for the speakers, we will appreciate that. The other housekeeping comment is that if you can come to the microphones and to ask the questions because otherwise this is a session that is recorded. So, it would not be captured very well if you don't speak through the microphone. The first speaker is Roger Bujona who has, he's an MD-PhD from the Catholic University of Leuven. He's an authority on vitamin D, has been working for decades on vitamin D and bringing seminal concepts and new discoveries in vitamin D. We have the privilege to have him today from Belgium to talk with us about vitamin D infections and immunity. Roger. Okay, thank you for, thank you chairpersons. I would like to have a look at my slides if possible. Thank you, thank you for being here and for asking me to talk about the favorite topic, vitamin D and health. There are a number of reasons to think that vitamin D may have extra skeletal effects and I summarized them here. The virtually universal presence of the vitamin D receptor, the one alpha hydroxylase in many tissues and above all the very large number of genes under the direct or indirect control of 125. And just to say it started already early in the vertebrates because not my experiments but from others. Zebrafish, 10 days old, an injection of 125 and 10%, 10% of the genes go up or down. So, and many of the genes are clusters including genes that are related to the immune system. Here you see a very primitive cartoon of the immune system. All the cells of the immune system can express the vitamin D receptor and some cells can with the appropriate stimuli produce 125 of the hydroxyl vitamin D. And that has an effect either on the natural immune system of macrophages or on the acquired immune system dealing with dendritic cells, T cells, B cells. I will start with the acquired immune system, highly specific, effective. That's vaccination is all about. That's what immunity against COVID is all about. And let's see what happens. Here you see again an incomplete overview. Dendritic cells are the start and once they are activated they present an antigen to CD4 cells that can then start to activate T helper 1, T17 cells with all the cytokines. And with the appropriate immune stimuli, they can produce 125 hydroxyl vitamin D. And that 125 hydroxyl has massive effects, especially on dendritic cells. The start of everything is also where 125 hydroxyl vitamin D changes even the morphology of the dendritic cells so that they are less antigen presenting cells with all the cell surface and cytokines involved. In addition, it inhibits the activity of the T helper 1 and the T helper 17 and their cytokines, so less aggressive. After a while, because the production of the autocline production of 125 takes 24, 48 hours. So it's not immediate. It is with some delay that it starts to taper down the aggressive part but upregulates the T regulatory cells and the T helper 2 cells. Okay. And then I will use one example of preclinical studies. That's type 1 diabetes. And then later on move to human data. The not mice is a typical example of type 1 diabetes with insulitis, peri-insulitis, and then insulitis and destruction. And one experiment I would like to show is this one. If not mice are kept in vitamin D deficiency for the first hundred days of life and then switched to a normal vitamin D supply and compare that with mice that have received vitamin D throughout life. What happens is in the control mice, the females have more type 1 diabetes than males but that's specific for not mice in general all over the world. But if they are D deficient, then the females have 50% more type 1 diabetes and the males have a doubling of the incidence of type 1 diabetes. That means that if these animals are D deficient early in life, there is some mechanism that predispose them to the genetic aspects of type 1 diabetes. And exactly the same data were published at about the same time from Hector de Lucas Laboratory by Margarita Cantona. Exactly the same data. And I always like to show at least one slide that is very recent. Last week, not mice given a normal chow diet or 400 or 800 international units a day. For the most, that's a lot. Normal, plain vitamin D. And you see if they receive a very high vitamin D intake, they have a much lower type 1 diabetes. So that's just the opposite of what you have seen with vitamin D deficiency early in life. And that is associated with an upregulation of T-regulatory cells and interleukin 10 CD4 cells, which are the regulatory cells. So it shifts the aggressive part to the less aggressive and more tolerance part of the immune system. And that is only a few examples. 125 or its analogs. And that has done in several laboratories can prevent or decrease the risk of type 1 diabetes in primary prevention before insulitis, secondary prevention after insulitis or after ILF transplantation. Yeah. And that's the example of type 1 diabetes. But you can also talk with other autoimmune diseases in animal models. It works in a very similar way. Is that also applicable for humans? That's, of course, the basic question. Munger did a very elegant study. She had access to the data of American recruits, military personnel. And, of course, at the time of recruitment, they stored blood sample. And they did have type 1 diabetes. But later on, 310 type 1 diabetes cases developed. And she compared the vitamin D status long before they had type 1 diabetes with controls. And as you can see, if they have a vitamin D status below 30 nanograms per milliliter, they have a much higher risk type 1 diabetes than the ones with a better vitamin D status. And if you exclude a few cases where the diagnosis is uncertain, it's even more significant. And that is also the finding of, again, Munger and U.S. military personnel now for multiple sclerosis. The ones at the time of recruitment have a low vitamin D status below 25 nanograms, have a much higher risk to develop multiple sclerosis later in life in comparison with the better ones. Of course, that is observational. Very large database. Very difficult to repeat, to give 7 million American recruits vitamin D or not. But at least it's an indication that that could be one of the many factors that regulate autoimmune diseases. So we need randomized control trials. And up to a few months ago, I would have said, there are no randomized control trials for autoimmune diseases. But the vital study came up with one secondary endpoint that I will show you in a moment. But more, even for me, more convincingly are Mendelian randomization studies. If you have genetic polymorphisms that predispose you to have a lower vitamin D status than all the others, and there are a few hundred polymorphisms that have an influence on serum 25 hypoxidin. Based on studies using 3 SNPs, 4 SNPs, 5 SNPs, or 20 SNPs, 5 out of 5 Mendelian randomization studies dealing with a massive number of data, patients with predisposed, genetically predisposed lower vitamin D status have a significant higher risk for multiple sclerosis again, again, again. And whether the multiple sclerosis starts early in life or in adulthood, it's the same. Five times out of five positive. For me, that's something you can never repeat with a randomized control trial. And it's probably early in life that vitamin D has an effect on the elimination of auto-reactive T cells in the thymus or other T cells that are auto-reactive that has an influence on the later development of autoimmune diseases. And now we have results of the many large randomized control trials, but there is only one that reported so far data on autoimmune diseases. That's the VITAL study from Johan Manson. And here, you see the results. If subjects, American adults, receive 2,000 international units of vitamin D, after four years, they have less autoimmune diseases than the controls. Either for confirmed autoimmune diseases or a much larger number of certain autoimmune diseases and possible autoimmune diseases. This is significant. This is just not significant, but the difference is the same. And of course, at that age, mean age, at the start of the trial is 67 years old. That means it's not type 1 diabetes and multiple sclerosis because they don't develop that at that age, but it's rheumatoid arthritis and polymyeliorheal rheumatica. But that's a good example of how the study, however complex it may be, should be done at a younger age to look at other autoimmune diseases. The natural immune system is exactly the opposite. Their 125-dialoxy vitamin D stimulates macrophages to defend yourself against bacteria and virus. And here you see an in vitro example. The burst capacity of vitamin D deficient animals is lower than controls and hemotaxis is significantly lower. And here you see a cartoon from John White. If a bacteria meets a cell, the receptors are activated and that starts bacterial killing by a complex set of mechanisms, including T helper 1, T70, and so on. That also stimulates the production of the vitamin D receptor and CYP27P1, the 1-alpha-hypoxylase, several folds, not a little bit, but four or five times higher than in the baseline situation. And that stimulates the production of catalysts. So in words, a monocyte meeting a mycobacterium tuberculosis, upregulation of vitamin D, 1-alpha-hydroxylase, 125-catalysts, and that has antibacterial effects. It can be blocked by a vitamin D receptor or mimicked by 125. Unfortunately, the best human trial was done in Mongolia. Children that are vitamin D deficient and have a high risk of tuberculosis. They receive vitamin D equivalent of 2,000 units per day for three years. No effect on latent or active tuberculosis. That's life. Let's look at other infections. And I will start with a study in Leuven where we looked at COPD exacerbations, which are inflammation. Population received 100,000 units of vitamin D per day. I wouldn't do that today. I would give it daily, but that was a number of years ago. But the time to first exacerbation was not different by vitamin D treatment. But in a pre-planned analysis, the ones that were severely D deficient had a much lower rate of exacerbations. First time of exacerbation or later exacerbations. But of course we have to look at all the studies dealing with vitamin D supplementation and infections. And as you can see here, a meta-analysis of Martineau, 20% lower rate of upper respiratory infection if subjects receive vitamin D supplementation. And most of the studies were dealing with population that were not perfectly vitamin D replete. They repeated that study just a year ago. And fortunately it's still significant, but the effect is not 20% anymore, but 8%. That's a viral infection. Because we are all interested, viral infection, flu, okay, COVID. Yeah, that's relevant. Does it have an effect on COVID? And I would like to spend a few slides on that relation. Essentially, a virus meeting a cell has the same reaction as a bacterial meeting a cell with active defense against the invader and also activating of the vitamin D receptor and the 1-alpha hydroxylase. In addition, the SARS virus activates complement and that generates a further activation of T helper one and trying to kill the virus. Also, additionally, upregulate vitamin D receptor and the 1-alpha hydroxylase produces catalysidin as you see there. But catalysidin binds to the angiotensin receptor two, which is the beating place for the spike virus and thus decreases the entry of the SARS virus in cells. Of course, due to the delayed production of 125, it tapers down the hyper reactivity of the system. And that is the major problem of SARS and COVID-19 because that's the reason why the immune system goes in overdrive and can cause acute respiratory distress syndrome. Human data. One first meta-analysis, subjects with a poor vitamin D status had a non-significant higher rate of infection. Small meta-analysis. A new one just also a few weeks ago in GCM. Yes, and you see how rapid the progress is in studies with vitamin D and COVID. Yes, if you are vitamin D deficient below 20 nanograms, you have a 46% higher rate of the incidence of COVID. So that's a question of infection, not severity. Let's look at severity. Oh, yes, no problem. Again, it's positive and then immediately there is a study to taper down my enthusiasm. A very nice study in the UK, special design of the study, which I cannot go into detail, but essentially a large number of British adults randomized to no treatment option. So it's not a classical placebo order. They were not even fully aware that they probably were the placebo group. But anyway, the others received an offer to measure 25-hydroxidine and most of them had levels below 30 nanograms and they received 800 or 3,200 units of vitamin D. And then, yes, what happens? Upper respiratory infection, non-significant. COVID-19 positivity, non-significant. Hospital admission for COVID, no difference. Yes. Then let's have a look at the severity of the disease. And here you see hospital admission or death. If you are de-deficient, based on this small meta-analysis, you have a higher risk of hospital admission or dying from COVID. Let's look at the interventions that they won in Brazil, giving patients that had COVID-19 received needed hospitalization, received a high dose of vitamin D, and then looked at the severity of the disease, and as you can see here, no difference in length of hospital admission or other major complications. And the same is true for the ones with a low baseline level of vitamin D. So that's a negative study. There was one pilot study in Cordoba, Spain, and some patients were randomized to receive a rather high dose of 25-hydroxy-D. So not just vitamin D, but 25-hydroxy-D, which immediately increases serum 25-hydroxy-D for a long time. And the others didn't receive calcifediol, but otherwise the treatment, of course, was the same. And here you see, of the ones that didn't receive calcifediol, 50% required admission to ICU, and in the others, one out of 50. So a massive difference in effect. Of course, it was the intention, a small pilot study in acute circumstances. That was the first wave of COVID-19, and Spain was extremely well, highly infected. And therefore, there was a second analysis of five hospitals in Andalusia. The majority didn't receive calcifediol. 70 other patients than in the previous pilot study received calcifediol. And you look at mortality, because now we are dealing with different hospitals, and maybe they have different admission rates to ICU, but mortality, that's the same in whatever hospital, I think. And you see there is a massive decrease in the risk of the odd ratio of dying if you receive calcifediol immediately after hospital admission. But it's not a proper randomized trial. Then there is one study, also very recent in Mexico, they looked at healthcare workers in front lines, so they are high risk of infection, and they received four dose international units of vitamin D for 30 days, and as you can see, the rate of infection was substantially lower in the ones that received vitamin D. So that's now a positive study again. The largest study, to my knowledge, is that in Barcelona. Patients were admitted to the hospital, the big hospital of Barcelona, and they didn't do a proper randomization, but they said, where is a bed available? Because it was an emergency situation. And they were either allocated to one of four wards, and all the patients in one of these four wards received calcifediol. And the others, based on availability of beds, were allocated to another ward, and they received no calcifediol. What happens? The ones that were lucky to be transferred to a ward where they received calcifediol, ICU admission, odd ratio, 0.13. Mortality, 50% lower. So these were the lucky ones. But technically, it's not a randomized trial on a person per person, but as a group according to the availability of beds. And here you see what happens. ICU admission after four days or so, five days, something like that, you see a difference in ICU admission, and you see it's a big, big difference, and also the mortality, that's a very big discrepancy between the ones that received calcifediol and the others. And then another type of clinical trials is what we call real world data. Andalusia, 6 million people, they have a very good healthcare record system. Overall, in that year, 2020, there were 16,000 patients that needed hospital admission because of COVID-19. And then they look back in the electronic database, and the ones that received calcifediol, a prescription 15 days before the hospital admission, and they were not sick for COVID 15 days before that, the mortality, because that's the hard end point, was one third lower. If they had received a prescription for vitamin D 15 days before admission, 25% lower mortality. If they looked at the database to the ones that received calcifediol or vitamin D 30 days before hospital admission, again, for calcifediol, 27%, lower mortality, and just not significant if it was vitamin D. So in summary, I don't see the timing here, but I think I'm running over time. I will be short. All cells of the immune system express vitamin D and react to 125. Animal data, I showed you one example of type 1 diabetes, but there are 50 or 100 publications or more that there is a relation between vitamin D status and autoimmune diseases, and the treatment can decrease the activity of the disease. Epidemiological data that I didn't have time to show, yes, poor vitamin D status, higher risk of infection or autoimmune diseases. I'm very impressed, as I mentioned already, by five Mendelian randomization studies dealing with 20 or 30,000 patients, that the ones that have genetically low vitamin D status have a higher risk of multiple sclerosis. Metoanalysis of Martineau that I summarized, if you receive vitamin D supplementation, a small risk of upper respiratory infection. It's not a massive effect, but at least it's consistent over two meta-analysis. I would say calcifediol, and that's the last slide that I showed you, seems to decrease especially the severity. ICU admission, acute respiratory distress syndrome that I didn't show you in detail, and mortality. But it's not based on large randomized controlled trials, but on something that comes close, but that's really the randomized controlled trial. And then the first ever trial for prevention of autoimmune diseases, not just for vitamin D, but for whatever compound. Vitamin D supplementation decreased the risk of autoimmune diseases in adults. And therefore, I would say, from a variety of data, it seems likely that the immune system is one of the targets of the vitamin D endocrine system outside the skeleton. Thank you for your attention. Thank you very much, Roger. Any questions to the audience? Low and clear because I have a hearing problem. I'm sorry. I'm from Brazil. Congratulations. I would like to ask you whether there may be an impairment on alpha hydroxyl activity during infections, particularly in COVID-19. So would you consider testing calcitriol for COVID instead of the calcifediol? So I mean the calcitriol? Yeah, that is a highly relevant question. But of course, I don't think that 25-hydroxyl-E by itself is active. It's neat. I know. I know. The point is, I conducted trials in COVID and we saw a very strong reduction in 125. So he found that the 125, the 1-alpha-hydroxylase is decreased during COVID. So the transformation in 125 inside the immune cells is decreased. So your question is, if you measure different... Actually, better now? I think so. Instead of giving calcifediol, if we gave in COVID-19, this is a hypothesis, not a... Calcitriol itself, the 125, because maybe there is a reduction in the 1-alpha-hydroxylase activity during COVID. And we just observed this strong reduction in 125 during the COVID itself. What do you think? In patients, I'm not sure that I have data on COVID infection, but in critical illness, there is a decreased production of 125-dihydroxyvitamin D in serum, probably in the kidney. Whether that also applies for the lung epithelium, I have no idea. I'm using 125, using 125 treatment instead of 25. Oh, yeah. I wouldn't do that because then you have a systemic effect. You don't want to have the systemic effect of 125, you want to have the local effect of 125 to avoid problems. I mean, if you don't have the study, I cannot give you a final answer, but I wouldn't do the study. Just a hypothesis. Okay, next. Thank you. Anne Owen from the Bergman Hospital, because I'm too old to be at the Erasmus Hospital in Brussels anymore. As you know, in Belgium, we have what I call the Mediterranean origin families, particularly Moroccans. And we also have many Congolese. Their vitamin D is almost always in the single digits. And it seems to be a hereditary thing. I've been trying to get the Belgian Frisbee study on bone density to separate these groups to look at them. And I wondered if you had any further information from your infectious studies. Because I wonder, sometimes groups with hereditary changes like that have managed to adapt. Do they really need all this replacement? I'd like to know for compliance. A bit too long for my ears. What is the question? What is the question? I was distracted. Just a short question. Is it a common question? Well, yeah, it comes to the COVID because the bone density studies haven't been done. So I wondered your experience with Belgian patients of these Mediterranean family origin. Have they adapted at all? Because their vitamin D, as you've probably noticed, is usually single digit, below 10. Yeah, I'm afraid that... So any comments about Mediterranean origin patients from Belgium in regards to immunity? Well, to immunity because they have low vitamin D levels. Yeah, I mean, Belgium is not that bad for vitamin D status. But the Spanish studies that I showed... These groups are... They have a rather low vitamin D status. Despite all the sun, that they have the mean level of 25-hydroxy-D in Cordoba. In that time of the year, that's 12 to 18 nanograms per milliliter. So it's low. The same is for Barcelona. You would say tourists go to Barcelona for the sun, but not all the Spaniards. They have a low vitamin D status. Do they have any resistance to the problems of low vitamin D since these groups have... Any resistance of any diseases because they have been for a long time with deficiencies. So if there is any accommodation... Of course, if vitamin D deficiency have other consequences for a lifetime, I mean, we will have two additional lectures. But that's a symposium on itself. What are the extra-skeletal effects of vitamin D? There was a hype a number of years ago that vitamin D would cure averaging. I think that hype has tapered down. But at least I show you some data that there are probably some extra-skeletal effects. But to a much lesser extent than what people believed a number of years ago that it could cure just every disease. I hope you understand what I mean. Yes. So we are going to move on with the next speaker. There are some questions online that we will not be able. Thank you, Roger. So I would like to welcome our second speaker, Lajos Kemeny from Semmelweis University in Budapest, Hungary. He's in Department of Dermatology. And he will be speaking on vitamin D deficiency, exacerbates, endorphin, and opioid addiction. Hi, everyone. Thank you very much for inviting me. I'm really humbled to speak in front of you. Being a dermatologist, it's not usual to attend endocrinology conferences, so I'm really glad that we'll get a little bit different feedback at this time. So as you may know, my title has to do with vitamin D and addiction and addictive behavior. I have no financial relationships to disclose. In case you have any questions, feel free to have a photo and submit those questions in the end as well. So in dermatology, what we were kind of puzzled about a few years ago is that there are a number of patients who suffer basically from substance abuse disorder by tanning and by tanning salons. So we have multiple random people who just seek tanning salons so often that actually they meet criteria for substance abuse. It has been explored a bit further as well. What do these patients feel when they go to tanning salons? And some of these patients can actually distinguish a real tanning bed from a mock tanning bed. It kind of means that these patients feel good, in fact, after they have experienced the sun, and it may have to do something with not the pigmentation itself but with the good feeling. And there was an interesting study when those patients who have actually healthy individuals who would seek tanning salons really often, when they were exposed to an opioid antagonist, naloxone, they actually exhibited symptoms similar to opioid withdrawal. So all of these kind of observations suggested that there is a behavior, an addictive behavior to UV radiation in humans as well. So this is why, as dermatologists, we became interested in addictive behavior a little bit. A couple of years ago there was a study from Mass General Hospital from David Fischer's lab that described that actually when it comes to addiction in mice, mechanistically it can be investigated really in depth, and the addictive behavior is due to the skin-derived endorphin synthesis. So how does this occur? After UV exposure hits keratinocytes, there is DNA damage. When it's large damage enough, then P53 will activate POMC transcriptionally. POMC is a well-known hormone that has multiple peptides in it. One of them is alpha-MSH. So alpha-MSH for us dermatologists is really important because it will activate MC1R on melanocytes, and it will trigger pigmentation in melanocytes. This pigment will be transported back to keratinocytes to further shield from further UV damage. So this is a really, really conserved pathway that has been conserved across multiple species in vector base as well. This is how even mice tend, just as we do humans. But then it was kind of interesting as well that POMC has endorphin encoded in it as well. So the obvious question was, does it have a function? Can endorphin levels go up high enough to have effects as well, or is it just like an epiphenomenon, a random effect that has no consequence at all? So this was investigated a couple of years ago in David Fisher's lab, and what they have found is that those mice that receive UV radiation, similar to a Florida 10-minute sunshine every day, they received those consecutively for several weeks. It was measurable that the pain thresholds went up in these animals. So it means that the androgen opioid signal was sufficiently robust enough to trigger analgesic effects in mice. Also, in the same study, it has been shown that those mice that have received chronic UV radiation for several weeks, and then they were subjected to a withdrawal assay by injecting them with an opioid antagonist, they actually suffer similar withdrawal symptoms as mice would who are opioid dependent. So all of these results collectively suggested that there is this addictive behavior in mice as well that is mechanistically linked back to beta endorphin. So why is this important? It's an interesting observation, but what is the problem with this? If you look at multiple cancer types, and if you rank them by mutation load, you can appreciate that the highest mutation is observed in melanoma, which we know, and also it has been described quite well, that it is linked to UV radiation-induced mutations. Quite interestingly, the second and third tumor types are lung cancer that are usually accumulating mutations quite heavily due to tobacco smoking. So it's kind of interesting that these high mutation load cancers have something in the background that has something to do with addictive behavior. So this doesn't sound to be a good effect. It doesn't sound, you know, a good idea in evolution to be addicted to one of the most ubiquitous carcinogens in the world, UV radiation. So we were wondering whether it could be an advantage. And then we came to the vitamin D field. As you probably all know, it has been hypothesized before that vitamin D has been the evolutionary driver for lighter pigmentation. So even in recent human evolution dated back tens of thousands of years ago, when people migrated to the north, it has been speculated that developing a lighter pigment tone might have been evolutionarily beneficial because there was less shielding. So there was more a chance to produce vitamin D. Also, there were several other evolutionary studies when they have traced back the selection dynamics for multiple genes that regulate pigmentation. And it has been found that in the northern latitude, multiple pigmented genes were selected for, whereas in high UV exposure regions, MC1R1 gene that has been shown to be associated with hair gene has been selected against. So all of these observations suggest that maybe vitamin D, even in recent human evolution, has been such a strong evolutionary driver that it can be traced back to lighter pigment tone. So we were wondering, what if this UV addiction pathway might also promote the same purpose, namely vitamin D production? It's really difficult to study evolution, especially using animal models. So we had this idea that maybe if there is an evolutionary feedback loop with vitamin D, it's quite common to have these negative feedbacks in evolution. So in the context of vitamin D deficiency, having an inclination to seek the sun more often could have beneficial effects by maximizing vitamin D synthesis, by maximizing the spent time under UV exposure. But when vitamin D levels are sufficiently high enough, and then where there's no further benefit for UV radiation, we would anticipate that a negative feedback should be turned on, and vitamin D should repress this addictive effect. So this is our proposed model that we were trying to study. But again, it's really difficult to study this. So we came up with an essay. And our question was whether vitamin D deficiency might sensitize directly to UV-mediated reward. So for this, we developed this essay. You may be familiar with the conditional place reference essays that is widely used in the addiction field. In case you're not, I'll just summarize it really briefly. So we place a mouse in a three-box compartment, and then we let the mouse explore all of these conditions freely. Then in a consecutive few couple of days, we place the different groups of mice in one container, in the black, for example. Then we have this neutral stimulus, which is a mock UV in this black box. And in the afternoons, using a group of mice, we will either use UV radiation or mock UV radiation. And then we'll place them into this white box. And then they'll spend 30 minutes here. So what happens at day 10? So after 10, actually after eight of these conditioning sessions, what we anticipate is that those mice that have received UV in a white box that previously they didn't like, they may have an inclination to that environment where they have experienced the good feeling. So this is one of the classic essays to model and study reinforcement behavior and addiction in mice. And then we develop this to UV. So using the conditions what we've established, using wild-type, VDR wild-type mice, mock UV or UV light, when it was used in a white box, didn't do anything. So in the post-test session, the mice couldn't care less whether they have experienced UV or mock UV previously. Similarly, when we use VDR knockout animals, when they have received mock UV radiation, they didn't care. They spend equal amount of time in the white box as before. However, those VDR knockout mice that have received UV radiation at a physiological dose, they have exhibited a significant preference to that white box where they have initially, they were neutral about, but now they kind of prefer this environment. So this suggested to us that in the absence of vitamin D signaling, the reinforcing rewarding effects of UV radiation is increased. So again, this is kind of an interesting thing, maybe for dermatologists, but like what is the therapeutic relevance for humans? So before COVID-19 in 2019, it was the first year when the life expectancy in the US was decreased and it has been attributed to opioid addiction. It has been risen over the last couple of decades. And then we wonder whether it is fancy what we have kind of described is that maybe vitamin D deficiency sensitizes to UV radiation and UV radiation reward. Maybe we could study this with morphine as well. Endorphin is an endogen opioid. So maybe these effects are not specific to this. Maybe a general morphine, heroin, all of these could have similar effects. So we tested the, we used a similar equipment. We used the CPP equipment, this three box apparatus as well. And then we did this dose saturation. As you can appreciate here, wild type vitamin D normal mice exhibited a place preference at a highest dose of morphine tested. But like in lower doses, five and 10 milligrams, they didn't really show any preference to that environment. In contrast, those mice that had no functioning vitamin D receptor had an at least fourfold shift in sensitivity to morphine. So this suggested that not only to UV reward effects, but also to generate morphine effects, vitamin D deficiency may somehow sensitize. So here we had also the advantage of using dietary vitamin D deficient models. When we used the VDR animals in the UV study, we didn't want to use dietary vitamin D models because UV may actually correct for vitamin D levels and this may just confound our experiments. But here we could do that. So we had mice that were a vitamin D deficient diet for two months at least. And then these mice actually phenocopied VDR knockout mice completely. So this suggested to us that not only the VDR knockout mice, but also the dietary vitamin D deficient model, dietary deficiency also sensitizes to opioid addiction in mice. Importantly, here we could also have an additional group of mice that were previously vitamin D deficient, but then we rescued vitamin D by adding vitamin D back to their diets. And this rescue was for two months. And what we could see is that the sensitivity actually restored to the levels of wild type animals. So this suggested to us that not only to UV reward, but also to morphine reward, a vitamin D deficiency may sensitize. And then we wanted to test for this a bit further and we tested other effects of opioids as well. So one of the most commonly used effects is the analgesia effect of opioids. And as you can appreciate in this figure, we have seen that both VDR knockout animals and both the vitamin D deficient animals have shown an increased analgesic response to morphine. So this suggested that not only rewarding effects are increased, but also morphine analgesia is increased in these animals. What's really important in the addiction field is the development of tolerance. So tolerance is defined by the loss of initial effects. So what we have done here is use VDR knockout and wild type animals and injected them with morphine every day and assayed for the effects of morphine. And what we have seen is over time, as you would expect, tolerance occurs in VDR wild type mice, but in VDR knockout mice, this tolerance was extremely fast. It was much more faster than the wild type mice. So this is one more endpoint of opioid addiction, which is more severe in a vitamin D deficient context. Lastly, what's really important is opioid dependence here. So this is what requires special attention because this is what we are trying to cure in the clinic as well. Those patients who have already developed opioid use disorder, this is what we would kind of model them in vivo if we can say that. So what we have done is we consecutively injected these mice with morphine at escalating doses for several days. And then after a week, we have subjected them to a morphine withdrawal assay, which means that we injected them with opioid antagonist. And this sudden gas station of the opioid signaling induces withdrawal symptoms. And as you can see here, we have actually assayed for multiple kinds of withdrawal signs. And in multiple withdrawal signs, we have seen an increased effect in the VDR knockout mice or vitamin D deficient mice. We also could like quantify this and summarize this. We could basically give them a morphine dependency score. And we have seen an increased dependency both in the VDR knockout and both in the vitamin D deficient animal models. And because all of these rely on subjective measures, we also had this body weight measurement as well when we could objectively measure the weight loss during this opioid withdrawal, which was also more severe in the vitamin D deficient context. So this was all interesting to us, but we wanted to have a mechanistic explanation for this. So what we did first is we injected mice, VDR knockout mice and wild type mice with morphine and isolated one of the key reward regions from the brain, glucose accumulants, and did RNA-seq. And actually we have found that CFOs is a common neuronal activation marker. CFOs activity has been predicted to be increased in the vitamin D deficient context. And then when we did a kinetic time course experiment, we also found that actually morphine induces a much more robust increase in CFOs activation in glucose accumulants. So we were kind of interested that maybe we could pinpoint it down to this region where vitamin D could have an effect. But then we looked at some transcriptomic databases and actually identified that VDR is very lowly expressed in this region in human brains. So it's unlikely that this would be the site of action. But as pointed out before as well, vitamin D is ubiquitously expressed in multiple cell types and multiple brain regions. So it's really difficult to find where it exactly has its effect. But still we did a bit more investigation and we focused on one of the upstream regions of glucose accumulants that gives projections to the glucose accumulants, the ventral tegmental area. And here what we have seen is that in the case of vitamin D deficiency, multiple genes that have to do something with dopamine metabolism were down-regulated. And this observation is a bit counterintuitive because we don't know why these mice would then react better to opioids. So it actually has been shown before that the vitamin D deficiency, I believe in rats, decreases dopamine turnover. And that's why rats that receive cocaine, they do not react as much as wild-type animals. So actually there's an inverse phenotype there. So this is a bit, it's a bit more complicated than someone could at first think. But here what we see is that a decreased dopamine turnover in the vitamin D deficient context, but still these mice experience a higher reward to opioids. So what we think happens here is that due to the constant down-regulation of dopamine signaling, when it comes to opioids, there's just an increased pleasure feeling due to the relatively higher dopamine increase compared to baseline. But this obviously requires further examination. So this is all mice. We wanted to move a bit further and study this in humans as well. So we turned to the NHATS dataset where there was some data available on healthy individuals on their vitamin D levels and also their self-reported opioid use. And what we have found is that vitamin D deficient, actually vitamin D levels were inversely and dose-dependently associated with self-reported opioid use. So this was something interesting because this was in line with our preclinical mouse data that vitamin D deficiency may somehow sensitize to multiple aspects of opioid addiction. And then also we wanted to look this a bit further to look at a population that actually suffers of opioid use disorder. So we looked at the MGH, internal clinical patient databases, and identified patients who had a diagnosis code of opioid use disorder and matched them to patients who actually had not had this diagnosis code. And they were matched by demographics and also principal care physician as well. And here we have also seen that vitamin D levels were inversely associated with the diagnosis code of opioid use disorder. So this collectively suggested that maybe in humans, we also see similar trends than what we could see in mice. So I just wanted to go back to our proposed model because this is one of the key take-home messages from our manuscript is that vitamin D deficiency may actually sensitize to UV addiction or UV seeking behavior in order to maximize its own synthesis. And our data is in line with a feedback loop where vitamin D levels are repleted. It's no longer requirement for this increased addictive behavior. Then this pathway is shut down. And in this regard, this could have evolutionary advantage of maximizing vitamin D synthesis, despite those negative consequences of skin cancer that I've shown you before. Also skin cancer usually develops at later ages, usually post-reproductively. So it may have less effects on survival and population fitness. And I also have a different summary slide as well that shows the consequences of vitamin D deficiency. So basically if vitamin D deficiency is prevalent in animals at least, either due to lack of UV radiation or lack of dietary vitamin D supplementation, these mice will have a deep repressed opioid signaling. So they will be much more sensitive to intrinsic, like UV-triggered endorphin, and also extrinsic morphine effects as well. And this could actually lead to an amplifying cycle of dependency as well to those that are at risk for developing opioid use disorder. So as a summary, I think I just mentioned this, that vitamin D may be an evolutionary driver for UV seeking behavior. It has been shown that it's associated with opioid use disorder by us using two different data sets. And since then, there has been one more study as well that have established that patients who have undergone major surgery, at a time of surgery, when they were vitamin D deficient, they had a much higher likelihood of developing opioid use disorder. And that was published just last year by Mariam Asghari from MGH as well. So we also think that we may have some therapeutic value here is that potentially modulating vitamin D signaling may have beneficial effects, well, first in opioid use disorder, but also potentially in other substance use disorders as well. It has been shown before that multiple other substances, like nicotine and alcohol, they all activate androgen opioids. So actually our data may not be restricted to opioids alone. And I'd like to thank all of our collaborators who also were a huge help in carrying out this interdisciplinary huge study. And I'll be happy to take any questions if you have any. Thank you. Thank you very much. Thank you. Thanks. Hello. Thanks for that. Dave Shuman, Cleveland Clinic, General Endocrinology. Is it possible that in those human observations that the narcotics those people were exposed to could be decreasing the vitamin D levels by increased clearance? I think it's hard to rule out. The third study I just mentioned at the end of my talk, I didn't have any figure about that. I think it may argue that it may not be the only explanation because those patients who have undergone surgery, they haven't had received any medications when the vitamin D levels were assessed. And when you fill them up, they had a higher increased likelihood of developing opioid addiction. So I think in that regard, so that suggests that there is something else as well. Maybe like our results. I can't rule out your explanation as well, of course. I have one question. How confident are you that the PMC promoter region contain P53 responsive element? So POMC has P53? No, the question is, how sure are you about this, that the P53 will contain, the PMC promoter will contain P53 responsive element? I think I'm pretty confident about that. There are several papers that have shown that in keratinocytes at least, P53 regulates POMC. And I think those were validated independently at least in two, three different studies as well. But I also, I think the lack of functioning P53 in keratinocytes ablates this response. I don't think that's necessary for the POMC at least in keratinocytes. But I think there are some other cell types where POMC may go up after UV radiation, but that's independent of P53. So there are other pathways as well, but dominantly when UV triggers POMC in keratinocytes is like a tenfold increase and that's mostly due to P53. Well, there is no agreement on the P53 involvement. And maybe there is an audience here which are endocrinologists and who work with the P2 et al. So maybe someone will look into the regulation of POMC if P53 is involved in the regulation of the POMC. Because the POMC in the skin is regulated through a very similar mechanism as it is regulated on the central level. There is definitely a cell type dependency as well. So I think it warrants investigations, but in melanocytes P53 doesn't seem to be a dominant regulator of POMC. Yeah, P53 plays a role, but I'm not sure. The original study which were repeated were done if we go in this direction on C57 Black 6 mouse. And then there is a PMC which supposed to regulate the melanin pigmentation. The mouse model on which the study was done is not correct because C57 Black 6 mouse is the agouti recessive. I took for the agouti recessive and therefore the melanin production is constitutive. PMC knockout mice produce L-melanin in C57 Black 6 mouse. Okay, we're gonna have a short question. Next one please. Frederick Serino, I'm very interested in your VDR knockout data. And in that the question I would raise is, is it possible that this is a vitamin D ligand independent function of VDR that is governing this? Because we know that the vitamin D can also possibly feedback to the VDR itself. So I would be wondering if this is a more of a VDR phenomenon than a vitamin D phenomenon. Let me ask to also add into that, that UV is causing the effect and UV produce multiple metabolites that are not just only vitamin D, right? So have you explored that, just following that? So, actually we did not. So in this study, we just based on the, expert protocols based on the previous study as well. And we assume that the effects are mainly due to endorphin. We have one, so we have observations as well that in the absence of endorphin in the VDR knockout mice, we do not see an increase in nociception, for example. So at least some of the opioid signals are definitely coming from that. And then regarding your question, we did not specifically test this, but I think all of our results point in the same direction that in the absence of vitamin D ligand or its receptor, we have the same effects. So I don't think that there's any argument that is going to say that there might be ligand dependent depression as well going on in the background. I think both have a similar field type at least. I wish I was surprising to some because there are some opposing effects when it comes to skin biology and pigmentation as well. But here, everything was pointing in the same direction. Yeah, and it would, but it could be, VDR itself could be the regulator because VDR is upregulated by the administration of the REN25 as well. Okay, well, thank you very much for your presentation. And we are for the next speaker. Our last speaker is Stephanie Sisley. She's an assistant professor at the Baylor College of Medicine, and she will be speaking on the regulation of weight and glucose homeostasis by vitamin D. Thank you so much. I know that I am the last thing between you and dinner, and so we will make sure that I finish on time. Those are my... Objectives. You. It will work. Yes. Like that. It will work. It will. Slowly. So I definitely would like to thank the organizers for inviting me to speak today and be able to show you the results of the last few years of work in my lab. My disclosures, QR code. So when I was a clinical endocrinology, a peds endocrinology fellow, there became all this data on vitamin D and that it was linked to everything. And interestingly for me, it was linked to the metabolic syndrome. So this is one particular paper, but there are really hundreds of papers out there that link low levels of vitamin D to pick your component of the metabolic syndrome. It's linked to diabetes. It's linked to lipids. It's linked to blood pressure. It's linked to obesity itself or fat mass gain. And so looking at just traditional vitamin D physiology, we know that you either ingest it or you make vitamin D. It's hydroxylated in the liver and then hydroxylated again in the kidney. And the 125, which has been brilliantly shown in the last couple of slides, is very active throughout many different tissues. We know that it has bone effects. We know it has effects on the parathyroid gland. We know it has effects on immunity that was shown previously. However, if you look at this slide, the pancreas stands out as maybe how vitamin D can be linked to diabetes. But there wasn't much evidence on giving vitamin D to humans that it actually increased insulin secretion. And so one of the questions was, how is vitamin D maybe linked? Is it the pancreas? Is it only the pancreas? Or is there maybe something else? And at the time, the lab that I was in was the lab of Drs. Randy Seeley and Darlene Sandoval. And they were studying the role of the brain on obesity and diabetes. And so we know that the brain is a really great regulator of glucose. And we know that the brain, specifically the hypothalamus, but other areas in the brain too, can sense different things that are going on in the body, can sense glucose levels, can sense lipid levels. And by sensing those things, it then will change the output to different organs. And so three organs that are very important for the regulation of diabetes would be the liver, where the brain can actually shut down the liver's production of glucose. It can change at the level of the pancreas how much insulin the pancreas secretes. And it can also change at the level of skeletal muscle how much glucose the muscle uptakes. However, we knew that from older studies that vitamin D could bind in the brain. But there weren't many studies really looking at what could vitamin D do in the brain. And specifically with diabetes, could vitamin D do anything in the hypothalamus to regulate glucose control? So several years back now, I happened to have some rats at my disposal. And so they had third ventricle cannulas in them. And so I gave 125 D3 into the third ventricle. And in an animal, the third ventricle is surrounded by the hypothalamus. So if you give something into the third ventricle, it's going to hit kind of the entire hypothalamus. And you can see here that when we gave the vitamin D and then did a glucose tolerance test, and this is an IP, so kind of similar to an IV glucose tolerance test in a human, the animals had improved glucose tolerance after just an acute dose of vitamin D. And that's also shown in the area under the curve. So this was exciting. It showed that at least vitamin D could pharmacologically do something in the brain to actually regulate glucose. And we had shown through other studies in this paper that this was not from leaking out of the brain and affecting the rest of the body. This was a specific effect in the brain. So we did this again where we gave vitamin D into the third ventricle and then looked at, well, where in the hypothalamus might it be acting? And I was a little bit surprised that the vitamin D caused CFOS, which actually you saw in the past talk, that vitamin D actually caused neuronal activation that we could see through the little brown dots on the screen there in the paraventricular hypothalamus, which is the kind of triangle that's dotted there. If we pre-treated the animals with a vitamin D receptor antagonist, we lost the effect, indicating that vitamin D seemed to be activating neurons in the PVH of the brain, and that effect was through the vitamin D receptor. Then thanks to Dr. Deanna Arbel, who helped me with this experiment, we gave vitamin D then directly into the PVH to say, okay, well, if it seems to be activating, what happens if we give the vitamin D not to the third ventricle, but instead we give it actually just directly into the PVH? And in this experiment, we gave it only on one side, so just unilaterally, and you can see that, again, vitamin D improved glucose tolerance in these animals. If we gave it to an animal where we had previously given them a lentivirus to knock down the vitamin D receptor, we lost the effect, indicating that vitamin D could improve glucose tolerance through the PVH, and that that effect was actually through the vitamin D receptor. However, that was a pharmacologic approach, and so there's no data whatsoever on what a endogenous level of 125 is in the brain, but I'm pretty sure the dose we gave was probably a pharmacologic dose. So we wanted to know, well, what does vitamin D do in the brain endogenously? And so to kind of get at that question, we decided to just look at what was the function of the vitamin D receptor in the PVH? And we chose two different models for this. We got, we received the homozygous floxed VDR mouse from Drs. Gardner and Chen at UCSF, and we decided to do two models. One model was we would actually give AAV-CRE bilaterally into the PVH of some mice. And by doing this, we first of all are able to delete the vitamin D receptor in an adult mouse, so you don't have developmental compensation, which was important, but obviously we're giving a virus. There could be off-target effects. We could, we have to, you know, deliver the virus, so there's potentially some damage that you're doing there. So we wanted to pair that with a genetic model, and so we mated these mice in a different experiment to a CYM1-CRE, and CYM1 is produced in the paraventricular hypothalamus as well as the amygdala. So this would allow us to genetically knock down the vitamin D receptor without doing anything to the animal at all. And we figured by both of these models together, if we got the same results, then we would feel pretty confident in what the vitamin D receptor does in the PVH. And so in the next few slides, I'm going to show you both models side by side and the results that we got. The virus model is in the blue and the pink, and the genetic knockout with the CYM1-CRE is in the green and the purple. So you can see that in both models, we get at least a 50% knockdown. So it's important in these models, unlike the Demay vitamin D receptor knockout mouse, we don't get 100% ablation of the vitamin D receptor. There is some vitamin D receptor there, but there's at least a 50% knockdown, and that was similar in both of the different models. In both models, if you lose the vitamin D receptor in an animal that has been on high fat diet for a while, you get impairment in glucose tolerance, specifically in males. So in both the models, you can see that the males had an impairment in glucose tolerance, and we see no effect whatsoever in the females. We interestingly saw no effect on body weight. This was against my original hypothesis, but I expected to see something on body weight because, a little bit, because the paraventricular hypothalamus is so important for body weight. It's where the melanocortin-4 receptors sit, and pretty much most of the things that we know about the paraventricular hypothalamus, they control body weight. But in both chow conditions and in high fat diet conditions, we don't see any effect of loss of the vitamin D receptor in the brain on body weight in either model. So it wasn't an effect of body weight. It also, interestingly, this doesn't occur in chow. So in an animal that is lean, we don't see an effect of the vitamin D receptor in the PBH on glucose. Now we had previously seen that vitamin D, exogenously, if you gave it into the brain, didn't affect glucose levels, but that seemed not surprising because their glucose levels were already normal, so you were trying to make them like super normal. But in these conditions, we kind of thought that maybe we would see something, and we don't. So there seems to be, and now we have three different models, where the fact that the animal is either, whether it's a diet-specific thing, because they were on a 45% high fat diet, or whether it was the obesity that was induced itself by the actual diet. There's something about that state in these animals that brings forth the effect of vitamin D, that either vitamin D can work exogenously, or that loss of the vitamin D receptor will actually cause impairment. So of course we want to know how, what is going on, and how is this happening. And so we just looked at the animals where we had just, through the genetic model, through the Sim 1 CRE model, where we had lost the vitamin D receptor, or knocked it down, and we just looked at the neuronal firing just at baseline. So we aren't doing anything to them, we just took the neurons out and said, kind of, how do they look? And so this was special thanks to Dr. Yanlin He. And so you can see that if you have lost the vitamin D receptor in the PBH, the neurons do not look nearly as active as the wild type littermates. And so to quantify that, if you look at firing frequency, the males that have knocked down of the vitamin D receptor in the PBH have a decrease in their firing frequency, as well as their resting membrane potential is more negative, which means that it's harder for the neuron to fire. You see that there's no effect in the females. And so this might be one of the ways that we're getting the sex specific effect in both of our models, is that there might be something that in female mice, they're able to compensate for the loss of the vitamin D receptor there. But there's definitely a sex specific difference in what vitamin D receptors do to neuronal activity. If you think about that, it looks like, right, since they're not firing as much, that maybe the vitamin D receptor is affecting an excitatory synapse. And excitatory synapses tend to be glutamatergic. And so we had previously done a study where we had given vitamin D into the brains of rats and looked at, did RNA-seq on the hypothalamus. So we kind of went back to that data and thought, did we see anything that was like glutamate related? And you can see that looking at that data, we saw three different genes that were important for glutamate receptors. We see the Grin 3A and the Grin 2A expression were increased. These are both components of the NMDA receptor. And then Grin 3 is an important component of kinase. So this was in rats and we gave vitamin D, but we definitely need to go back and look at our mouse models to determine whether or not we see any differences in the expression of these receptors in mice where we have lost the vitamin D receptor. But it's intriguing. So then we wanted to know, what does vitamin D do to these neurons? I showed you what the loss of the vitamin D receptor does, but we wanted to know what does vitamin D itself do to these neurons? And so with a collaborator, we gave vitamin D to a neuron and you can see that they are activated. I'm going to point out here that about between 70 and 75% of the neurons were activated, but there is this kind of 25% or so that are not activated. Remember that in this model, we are actually pulling neurons based on their fluorescence, which is based on Sim 1, not on the vitamin D receptor. So in order to actually look at these neurons, we're just picking up neurons that are in the PDH, but we don't know whether or not they have the vitamin D receptor in the neuron or not. And I wouldn't expect that there's vitamin D receptors in every single neuron. So you can see that in males and in females, actually, neurons can respond to vitamin D. And what you see on the right is actually that the effect of vitamin D on the firing frequency, the delta, was no different between the sexes. So the sexes are not responding to vitamin D differently, and they can both respond to vitamin D. And both of their response is due to the vitamin D receptor because you lose that effect in the animals that have knockdown, which is the KD. We see something very similar with the resting membrane potential. So again, vitamin D can actually improve that, making it easier for a neuron to fire. It is through the vitamin D receptor because you lose the effect if the animal doesn't have the vitamin D receptor, and it can do it in both males and females. So what are the vitamin D receptor neurons? So I kind of hinted at this might be a novel population. Years ago, we tried to use immunohistochemistry, and we used two very well-known antibodies. And we did this in a wild-type mice and the domain knockout mouse. And so there is no vitamin D receptor in that knockout mouse. They are small. They are hairless. They are hypocalcemic. There's no question. And yet, I can't tell a difference in these slides. The antibodies, at least in the brain, while they are specific in other tissues, at least in the brain, they are not specific. And so this actually hampered the research quite a bit because we couldn't use immunohistochemistry to kind of identify the neurons in the PDH. So we created a new mouse. We created a reporter line. So we did this by creating a VDR CRE mouse, where the CRE recombinase is expressed in cells based on the expression of the vitamin D receptor. And then we mated that to a reporter line that was a CRE-dependent tomato expression. And so you can see here that there is vitamin D receptor expression in the PDH as indicated by tomato fluorescence. But we needed to validate that. So to validate that, we looked at a neuron that is expressing tomato. And we gave vitamin D, and you can see that it lights up, or it's activated. If you give vitamin D to a neuron that did not have tomato fluorescence, there's no action at all. And if you gave vehicle to a tomato-positive neuron, there was no action, meaning that there was specificity of the vitamin D. So when we look at these neurons, specifically where we can target a vitamin D receptor neuron and we give vitamin D, we see very similar to what we saw before, except for now it's 100%. So 100% of our vitamin D receptor-containing neurons actually had improvements and increases in their firing frequency, and that did not occur in the neurons that did not have tomato. And that was the same in males and females. So we did two additional studies to look at what were these neurons. And so we looked at vasopressin, so using the reporter, our fluorescence, and then we used antibodies for vasopressin. You can see that there's a few cells that are co-localized, no difference between the sexes. And then we looked at oxytocin. And there's a few more cells that are co-localized, but you can see there's a lot of cells that are red there that are not, do not co-localize with oxytocin. So we're kind of left with, we have a few of these cells are vasopressin, a few more are oxytocin, and there's a bunch that we don't know what they are. And so one of our next steps is definitely to define these a little bit more. So to summarize, vitamin D can act in the brain to improve glucose tolerance exogenously. Vitamin D receptors are required for glucose tolerance in males, and they are also required for normal neuronal activity in males. But the function of the vitamin D receptor in the PVH is impacted both by sex and by diet. And so we have a lot of next steps. We want to define the function of these neurons. So what do these neurons do? Where do they go? And thankfully this work was just funded through the NIH through an R01, so we were excited to get on that. We want to determine the effect of peripheral vitamin D. So I did not touch at all on all the clinical trials that have occurred with diabetes and vitamin D. The bottom line is there's nothing that is consistent. So there's a few that maybe show something, and there's a lot that don't. It's pretty sad that we cannot look to vitamin D to be this cure-all for diabetes. Vitamin D is very complicated. So if the brain is important not only in mice and rats, but also in humans, we give ergo or cholecalciferol. It requires two hydroxylation steps. It has to get across a blood-brain barrier, and I already showed you that there's differences between if you have an obese state or not as to how vitamin D works. So there's a lot of different steps there that would be maybe why a clinical trial isn't working as well. And so we want to look at the effects of different peripheral analogs, the effect of different diets, and see whether or not if you give a peripheral analog, do these brain neurons light up? And now that we have the VDRcree mouse, we can actually do that. We want to define the cells a little bit better, and then we obviously want to understand the sex-specific differences better. So is estrogen something that, because vitamin D receptor and an estrogen receptor are both nuclear hormones, is it possible that estrogen, if you have estrogen receptors around and they're activated, more so in females, that there's like compensatory things that can happen in a female that maybe aren't happening in a male? And so when you pull the vitamin D receptor action away, right, you see the effects more in a male. That's the current hypothesis, but we clearly want to test that more. I have many acknowledgments, many collaborators that have helped me along the way, and obviously the work couldn't be done without the funding through the USDA and the American Diabetes Association. And with that, I'm happy to take questions. Thank you very much. I got a question while the questions come. Oh, there are too many, so I don't want to do... That's probably dinner. Oh, maybe. So one of the questions could be that during evolution, you generate knockout mice that during embryogenesis they adapt, as well as you do CRE models, which I don't know the specifics in one, they start expressing very early in life. So it's potentially that you may not see the difference, depending on that there is an adaptations in the CNS that they were embryogenically adapted to the circumstance. That is one. And the second potentially is there is certainly the neurons in the area of the third ventricle around the third ventricle are affected by many inflammatory cells that are around it. Did you find anything on those specific knockouts or not? So I'll address your second question first. Regarding inflammation in the brain, because clearly, I will agree, to me that seems like the most likely hypothesis as to how or why vitamin D works in an obese state versus a lean. We have not been able to, partially because we just got the VDR-CRE mouse, and so prior to that we couldn't stain for different things, and so we were kind of truthfully hampered by that. We have done a few studies where we try to look at expression levels of TLR4 or different kind of inflammatory markers in the animals where we gave vitamin D, and we didn't see an effect. But the problem is in those particular studies, they're complicated by the fact that I'm literally inserting something in, and so I'm not sure we could see the effect well because we're probably causing inflammation just by the way we did it. We have not gone back yet to the mouse models where I didn't, at least the Simlin-CRE where we didn't insert anything to see if there was a different inflammation, and we have not been able to yet do like looking at slides with immunohistochemistry to stain for other markers. But that certainly would be a great thing to do. Regarding your first question with the evolutionary biology, I'm not sure. That was one of the reasons why we did two different models to look at the effect because we didn't want to have developmental things, but as far as how vitamin D acts or maybe the sex-specific effects, I mean, maybe. Right. Or you can generate an inducible trait that you can really postulate that this is not a developmental defect, and that is what you can potentially do. Yeah. All right. Any other questions about this interesting topic? Thank you so much. Things like the journal. Thank you.

vitamin D

addiction

weight regulation

glucose homeostasis

UV radiation addiction

opioid addiction

vitamin D deficiency

addictive behavior

glucose control

hypothalamus

neuronal activity

diabetes management