Hello everyone, welcome to the session on Molecular Mechanisms Controlling Energy Balance and the Development of Obesity. My name is Lawrence Kazak from McGill University, I'll be moderating this session and today we have six speakers. Each talk is ten minutes in length with about five minutes for questions. So I'll call up the first speaker today, which is Ron Yee from the University of Laval. I haven't seen Ron so I don't know if he's here. Maybe we can go to the second speaker and then if Ron shows up he can go last. So Wen-En. Wen-En is from the University of Illinois at Urbana. Wen-En. Wen-En Zou. Thank you to the Endocrine Society and the session organizers for giving me this opportunity to present my work here. So today I will talk about Pharynzoid X-Receptor, FXR, regulates the expansion and metabolic function of adipose tissue upon caloric excess. The prevalence of obesity is alarming and affects two-thirds of the adults in the U.S. Obesity is a major risk factor for developing type 2 diabetes, cardiovascular disease, and cancer and is associated with dysregulated lipid and glucose metabolism. Adipose tissue plays important roles in regulating whole body energy homeostasis. There are three types of adipose tissues, white, brown, and beige. Adipose tissue stores energy, release fuels for other tissues, secret hormones, and inflammatory molecules. Brown and beige adipocytes, they are rich in mitochondrial and specialized to produce heat during cold exposure. So adipocytes undergo rapid and reversible morphologic changes in the transition between fasting and the fat states. Once the expansion limit is reached, adipocytes is associated with hypertrophic, chronic overnutrition or obesity will lead to hypertrophic adipocytes, which is accompanied by inflammation and metabolic dysfunction. FXR is a nuclear receptor that transcriptionally regulates bioassays, lipid, and glucose metabolism in the liver. FXR is expressed in many tissues, such as the liver, small intestine, kidney, adrenal gland, and white adipose tissue. Although the role of FXR in liver-gut axis has been shown, its role in adipose is still poorly understood. It is known that FXR is expressed in white adipocytes. Adipose FXR has been studied in cell cultures and ectopic expression mouse models, but it is still unknown if FXR is expressed in brown fat and the exact role of FXR within the adipocytes is still poorly understood. So my objective is to define the role of FXR in adipose tissue remodeling during obesity. So first, we measured the expression of FXR in mature adipocytes isolated from different fat depots, and we found that the mRNA levels of FXR is high in gonadal white fat, but low in brown and subcutaneous white fat. Then to address if there is a direct role of adipose FXR, we isolated pre-adipocytes from white fat and differentiated them in the presence or absence of FXR agonist GW4064, and examined the expression of genes regulating lipid metabolism, which are known targets of FXR in the liver. And we found that activation of FXR elicits similar effects between the adipocytes and the liver, such that activation of FXR reduces SREBP1C mRNA levels and increases CBT1-alpha and PPAL-alpha expression during adipocyte differentiation. So next, to study adipose FXR in vivo, we generated the adipocyte-specific FXR knockout mice by crossing adiponectin CRE with FLOX FXR mice, which were obtained from Dr. Christina Skunia's lab. So the knockout mice shown in red show the robust reductions of FXR mRNA levels in mature adipocytes isolated from both brown and the white adipose tissues. Then we fed the knockout and the FLOX FXR control mice with either a high-fat diet, western diet, or chow for four weeks to study FXR's role during obesity. We first capsulized their body weight, fat mass, food intake, and energy expenditure. So the knockout mice and the control mice exhibit a similar body weight gain and the fat mass, except for the brown fat, which weighed higher in the western diet by the knockout mice than the control mice. So there is no difference in the food intake and energy expenditure between genotypes when these mice were fed either a chow or high fat. So then we look at the adipose morphology for the white fat. The knockout mice shown in the bottom, they displayed adipocyte hypertrophy, even under normal chow conditions, and this effect is more pronounced under high-fat diet. Similarly for the brown fat, the knockout mice also exhibit adipocyte hypertrophy, which is pronounced under western diet conditions. So since adipocyte hypertrophy is associated with insulin resistance, we examined the systemic glucose homeostasis by performing glucose-tolerant tests, the epiGTT. So the knockout mice shown in the red, control mice shown in the black. So under chow conditions, there is no difference for the glucose-handling ability between genotypes. However, under high-fat diet conditions, the knockout mice exhibit glucose intolerance. Similarly, under western diet conditions, the knockout mice also exhibit glucose intolerance. Since adipocyte hypertrophy is also associated with fatty liver disease, so we look at the liver morphology. So under chow conditions, there is no difference. However, under high-fat and western diet conditions, the knockout mice exhibit increased fat accumulation in the liver. So finally, to gain a broader view of the transcriptional regulation of adipose metabolism by FXR, we performed RNA-seq analysis on the white fat from the knockout and control mice. So you can see under chow conditions, the knockout mice exhibit reductions in metabolic pathways, including lipid and glucose metabolism. But under high-fat diet conditions, the knockout mice exhibit decreases in the immune response and the increase in the lipid metabolism. So this data show that the knockout mice displayed distinctly diet-specific transcriptomic changes. So overall, our findings show that adipocyte FXR is dispensable for food intake and energy expenditure. Adipocyte FXR can regulate adipocyte size, glucose homeostasis, fat accumulation in the liver, and the transcription of genes involved in lipid metabolism and the immune response. Hopefully, our findings can convince you that the loss of adipocyte FXR has a physiological consequence. So I would like to thank my mentor, Dr. Sayeed Anak, and the entire Anak Lab, as well as our collaborators, Dr. Cecilia Liu, Kevin Kim from UIUC, and Dr. Kevin Rosen from Howard. I would also thank the fundings. Thank you. I would like to take questions. Thanks, William. We have time for questions. Go ahead. Aaron Sipas, NIDDK-NIH. Great to see that you're working on this project. I want to know if you've been looking at the bile acid synthesis enzymes or aspects of their production or release into the circulation from the white or brown adipocytes. Yes, that's a great question. So first, we measured the circulating bile acid levels, and then we found that the knock-on mice showed reductions of circulating total bile acids under high-fat diet conditions. But under normal chow and western diet, there's no difference. Further, we also characterized the adipose bile acids. We found different bile acid conversations between the knockout and control mice in both brown fat and gonadal white fat. But for the bile acid levels within the adipose tissue, there is no difference. Christy Brownwell-Carnell. Very nice work. I might have missed this at the beginning, but I presume these are male mice, and I wonder whether you've done the study in both sexes. Sorry, can you— Was the study done in male mice? Yeah. And have you looked at the phenotype in females? Okay. So we performed a study in both sexes. So right now, all the data shows it's in males. So for females, there is less pronounced effects. For example, the glucose tolerance. So females, there is not significant. But we can still see a trend. Thank you. Nick Webster, San Diego. So I was really surprised by how little hepatic steatosis there was in the control animals. So what was the composition of the high-fat diet and the Western diet, and how long did you treat the animals? We fed the mice only for four weeks. So we would like to study the early onset of the obesity. But for our ongoing studies, we also feed the mice for a longer time, like eight weeks to 12 weeks. And is it the 60% high-fat diet? Yeah, 60% high-fat diet. And the Western diet is at 0.2 ppm glucose tolerance? About 5%, and also high sucrose. High sucrose. Yeah. Thank you. Maybe I can ask a question. You mentioned that there weren't any changes in energy expenditure, but that was just in the basal state. I'm wondering if you tried to challenge the mice with like a beta-3 agonist or a cold exposure to try to reveal any phenotype? Yeah, that's a great question. So we have challenged the mice under 16 degree, a mild cold exposure, but we cannot see any difference. So probably in the future, we should treat them with the 40 degree to have some acute exposure, cold exposure. Okay, great. Let's move on. Thank you. Thank you very much. The next speaker is Tiffany Miles from the University of Arkansas. All right. I'm Tiffany Miles. I'm a postdoc at the University of Arkansas in the labs of Dr. Gwynne Childs and Angus McNichol. And I thank the organizers for allowing me to give this oral presentation on one of my projects, which is looking at the effect of maternal undernutrition on the male offspring's resistance to high-fat diet-induced weight gain. I have no financial disclosures. So as we all know, there is an obesity crisis in the United States, and Arkansas, which is where we are housed and researched, is ranked number three in the nation. This is compounded by the fact that 42% of the population lives in food deserts, and Feeding America reports that one in four are Kansans struggle with hunger. So it's imperative that we study not also overnutrition, but different forms of malnutrition to see the effect that this has on this population, whether it's deficiency, excess, or an imbalance of the nutrition. Leptin, as you all know, is secreted by adipose tissue and proportional to the abundance of the adipose levels, and it binds to receptors throughout the body, and especially also in the pituitary gland, which is what our lab studies, pituitary function. And we have shown that leptin signaling is imperative to somatotrope function. This work in our lab used leptin receptor knockout mice, where leptin was specifically ablated in somatotropes, where growth hormone is secreted, and they show that there was fewer cells that was expressing the growth hormone protein. These mice developed metabolic dysfunction in adulthood. They had adult onset growth hormone deficiency and obesity, and they had a tendency for weight gain, seen as early as in neonatal development. Another concept I want to introduce to you all is the thrifty phenotype. So there is thoughts, and actually some studies that support this, that leptin signaling is regulated by maternal nutrition, and that when this leptin signaling is dysregulated, it can lead to metabolic dysfunction in the offspring in adulthood. And so I want to show you a study that was done in 1998 by Hema et al, in which they characterized the neonatal leptin curve. This was done in C57BL6 mice, the female offspring of normally fed dams. And they showed, let me see if I can get the pointer to work, here we go, OK, here we go. They showed that the leptin surge peaked at postnatal day 10, and it was independent of the adipose levels of those mice. So they were seeing that surge independent of adiposity, and there was no correlation. And so at this time, and then and even now, we do not know exactly what causes the surge. And later work wanted to, you know, manipulate that surge and see the effect of maternal nutrition on it. So they did a 50% undernutrition. These were in male rat pups. And they showed that with that 50% undernutrition that that surge was actually ablated, which is what the white bars. Another study did a 30% undernutrition in a C57 mice, and they combined the data of the male and female offspring. And they showed that there was a premature surge that occurred about postnatal day seven before the fed offspring. So we're seeing that the severity of the maternal undernutrition affects not only the timing of that leptin surge, but also the abundance of it. So this led to the study that I'm doing now. We wanted to determine how the maternal undernutrition contributes to offspring metabolic dysfunction. And how the loss of the perinatal leptin signaling induced by the maternal nutrition affects offspring's hematotrope function. And in my study, I used FEB mice. We have, we use FEB mice in our lab because we have genetic mice models. And FEB are more prolific, giving us more numbers in our genetic mice models. So we wanted to compare that, this strand to our other genetic models, which we did the leptin receptor knockout. At Proestrus, we mated our mice. We determined vaginal plugs at day zero. Ten days after mating, I measured the mice, the females, to make sure that they had gained weight and that they were pregnant. And I started the maternal undernutrition at embryonic day 15, which is late gestation. With our mice, I initially did a 50% undernutrition, but we found that that was too restrictive. Our mice are a little bit hyper than the C57s. So I had to drop it down to a 20% undernutrition. And then the pups were born at embryonic day 19. We collected pups at postnatal day 5, 8, 11, and 16. So that can characterize a neonatal leptin curve with a 20% undernutrition. As you can see on the graph on the left, the weights of those mice were significantly lower than those, the weights of the undernourished mice were significantly lower than those from the fed moms. And we had an interesting finding. That leptin surge, this is in the blue bar, of the undernourished offspring occurred earlier than those of the controlled fed moms. And there was higher as well. So we actually were able to show a premature leptin surge with our 20% undernutrition model, which was similar to the 30% that was published. We also had dysregulation of the IGF-FGH axis, in which IGF-1 was decreased in these undernourished offspring as well. Data that I'm not showing in this presentation was that we had delayed puberty in females by the presence of a vaginal opening, and that the males did not catch up in weight to the fed offspring. It took about seven weeks for them to catch up in weight. So now that we had our model, we, like I said, wanted to use a natural method to disrupt leptin signaling to the pituitary. We wanted to see now, as these offspring were grown to adulthood, how they would respond to stressors such as a high-fat diet. So we generated more pups, allowed them to grow to adulthood, and at 10 weeks of age, I did two cohorts, at 10 weeks of age, I'm sorry, 12 weeks, no, I'm sorry, eight weeks of age, and 12 weeks of age, I started them on a high-fat diet. And this was a 45% high-fat diet. So the first cohort, as I mentioned, started at eight weeks of age, and we have the weights for the males and the females. Interestingly, we saw that the males did not gain weight on the high-fat diet from the undernourished moms. Females, it was kind of hard to see exactly, because we did not have our control females gaining weight on the high-fat diet, so it was kind of hard to see what was going on here. So that's why we repeated the cohort, in which we started them on the diet at 12 weeks of age, so a little bit longer, and then we fed them for 16 weeks on the diet. And we were able to replicate in the original cohort the findings that we saw in the males, and the females, both from the undernourished and the fed dams, gained weight on the high-fat diet. So we're seeing a sex difference now to the response to high-fat diet feeding from undernourished dams. So next we want to determine a mechanism. We're seeing a phenotype in the males that's different from the females, so we wanted to see, okay, what is contributing to this lack of weight gain to the high-fat diet feeding? So we did bulk RNA sequencing, only on the males at this time, because of the restrictions at the time, but we only did it on the males, and we said, whatever findings we find in the males, then we'll measure that in the females. So we had six pups, or six offspring per condition, submitted pituitary, liver, and fat for RNA sequencing. As I mentioned, we're a pituitary lab, so that's our first tissue that we're interested in, and what we found, for each one of these comparisons, I'm gonna show you the difference between the controls, so the undernourished and the fed controls, and then the differences between the high-fat diet and the control for undernourished and then for the fed. So just comparing the controls, we say that there's a little bit of difference just between the controls, despite having been fed on a high-fat diet, so the pituitary is already being programmed differently, before they even are induced with high-fat diet feeding. And then, with a high-fat diet from the fed offspring and the undernourished offspring, we do see changes in genotype expression. One gene that I was curious about is TESPO1, which is downregulated in the undernourished when comparing with the controls, but then with high-fat diet, feeding is completely reversed. Then we looked at the liver. The livers were different with the controls, so there's pre-programming already there, but with high-fat diet feeding, we're seeing that they're responding the same with a high-fat diet, regardless of the maternal nutrition, and that the genes that are involved are, let me show the pathway analysis, are all involved in NASH. So we can see that they have infiltration or inflammation signaling, or infiltration of macrophages. Next, we wanted to look at the fat, because that's where we were noticing, phenotypically, that these mice were not getting away on the high-fat diet. Controls did not seem that much different from one another, but when we compared, based on the high-fat diet feeding, we're seeing that from the fed offspring, there's drastic differences in genes that are expressed. A lot of them involved with inflammation, whereas those from the undernourished moms, we only see about three, there were three genes that were specific to the fat of the undernourished offspring that were not seen in any other tissue or any other condition, so we're very interested in those. And then, here's a pathway analysis through ingenuity pathway analysis, and just kind of want to show you how those genes correlated with one another, and as you can see, they're all, a lot of them involved with inflammation, signaling, and cytokines. I did not have pathway analysis for the undernourished, because there was not enough genes to actually do a pathway analysis. And then, these were some of the canonical pathways that were activated or deactivated with the high-fat diet. So, to summarize the results, we have used a 20% undernutrition, maternal undernutrition model, which has skewed the neonatal leptin surge by causing it to become premature. Our offspring, the offspring from these moms are underweight. Growth hormone, which I did not show you in this presentation, was actually elevated at postnatal day one. IGF-1 was reduced at postnatal day 18, and when these offspring were, as adults, were given a high-fat diet, we saw weight gain in the fed and the undernourished for the females, but only the males from the undernourished moms did not gain weight, and we saw leptin was actually increased in the fed males on the high-fat diet, but was not seen in the females. And then, when we did bulk RNA sequencing analysis, the pituitaries were pretty much changed, a little bit, the same number of genes were pretty much changed for both the underfed and fed, though the controls were different. Liver were changed as they both responded to high-fat diet in the same manner, but what we saw a drastic difference was in the fat of the fed offspring. They responded more to the high-fat diet than those of the undernourished. So, by understanding these mechanisms, we can better combat this prevalence of obesity, looking at markers that may give us clues as to how we can, like, to diet, not diagnose, but to actually help populations to better understand the mechanism or what's going on in their population with obesity prevalence. Thank you. Sorry. I'll take any questions. Oh, and I forgot to say this. This is my lab. We're a collaborative team. Dr. Gwen Childs, Dr. Angus McNichol, and then Dr. Angela Odo, and we have several grants from NICHD as well as NIDDK. Several of our students are presenting. I can't believe I forgot that. Please go visit their posters today at 12.30, and we have one, Jutan is actually giving a rapid-fire presentation. So, please stay. Thank you all. Okay. So, we're looking for studies that show that maternal malnutrition leads actually to an increased risk in obesity and diabetes. How can you explain the difference in the males in your study? Is this a result of the 20% food reduction versus, you know, larger percent? Correct, right. Yes. So, that's what we were hoping for. We actually thought we were gonna have an increase in high-fat diet, more so than from the fed offspring. The 30% under nutrition paper that I just mentioned, they saw an increase in weight on the high-fat diet, and they even gave exogenous leptin at an earlier time point, at that PND5, and saw that increase in weight gain on a high-fat diet. So, it was new to us to see that those males were actually, did not gain weight on a high-fat diet, and that's why we repeated it twice, to confirm our findings. But it does, I believe it will correlate to the original observation in humans from the Dutch Hunger Winter, in which they show that moms who are undernourished in late gestation, actually their offspring, the male offspring, didn't gain weight on a high-fat diet, versus moms who were undernourished in early gestation, and their males gained weight on the high-fat diet. So, that's probably where it is. Timing of when the undernourishment took place, as well as the severity of it, is not as severe. Great, thanks. And a quick follow-up, do you see a transgenerational effect as well? I wanna do that, but I haven't done it, yes. Thank you. Hi, Tiffany. Hi. Nice talk, congratulations. This is Hongxia Ren from Indiana University, School of Medicine. I have a quick question. So, ask your comments or perspective about measuring the neuropeptide levels, the protein levels in the pituitary. It's nice you see the transcriptional changes in the pituitary. Any comment? I have not finished measuring all of the hormones in the pituitary. The protein levels did measure GH in the serum. Which was shown to be decreased with the undernourishment. But I did not finish measuring. So, that is something that I'm gonna do, actually measure those proteins. Thank you. Thank you. Very fascinating talk, lovely presentation. So, I was just fascinated by that leptin surge you showed me. So, and you know, based on that conflict because of the Dutch famine, is the surge changed depending on if they are undernourished early versus late? And have you looked at food intake in these ones? Okay, so is the surge changed with undernourished versus late? So, yes, from what we're seeing, oh, versus first trimester versus late trimester. I don't know if that's been shown in humans, the change in that surge. Because the surge is supposed to happen a third trimester in humans, whereas postnatally in rodents. It's suspected it's happening first and third trimester in humans. But measuring that actual surge due to undernutrition, I'm not sure if that's done and I'll have to look into that, yeah. Or at least if you can do it in the mouse to kind of resolve some of the conflicts. Yes, yes. And food intake, did you look at the opposite hormone, the ghrelin levels in your mouse? No, we didn't. That is something that we do look at. I did look at growth hormone secretagogue receptors, so the ghrelin receptor. I didn't see any changes with that, yeah. Yes. Very nice work. You talked a few times about the timing of the diet and the impact. What about at the time of lactation? And so I guess I have a technical question with regards to whether you keep your mice on diet during the lactation phase. And then more of a societal question with regards to individuals in food deserts. Is the prevalence of breastfeeding higher or lower in those populations? Okay, very good questions. So they were undernourished starting at embryonic 15 until those pups were sacked. So it could have been PND 5, 8, 10. When the offspring were grown to adulthood, they were undernourished until they were weaned. So all the way through lactation. As far as the prevalence of this in this specific population, I am making connections now with public health workers to get the data from those communities. We don't know, from right now, I don't know that. So I will ask. Yes, thank you. Carlos Bernal from Washington University. Great talk and a very important subject. My question is regarding of you do the changes in nutrition in the late pregnancy. Were changes in the placenta, were changes at birth? So do you then determine or do you show us if there was a determined changes in the size of the animals at birth and if that has an impact? As well as if you use foster mothers because you can make a foster mothers to change the implication of postnatal, particularly in the first weeks of life. Thank you for those questions. No, we did not measure the size at birth for one reason. I know personally for my reason, these FEB are very hyper. When I did the 50% under nutrition, they were eating the pups. So I did not want to mess with them while we were doing the 20%. But I did count how many were there, but I did not measure their sizes at that time. I only measured it if I sacked the offspring and just saw that at P and D one, they were smaller than they were at the, yeah, compared to the fed. Yes. And sorry, the next question. I missed that. Oh, foster. So from what I understand, correct me when I've read papers like that, please correct me. They fed the moms all the same ad libitum and then they switch the pups and then did either larger pup size, litter sizes or smaller litter sizes to the nutrition. So that would have been after birth. And we were trying to see if we can do changes with the under nutrition during gestation, before the hit and net surge. Yeah. I understood that if the mothers are under nutrition, they're weaning, right? So your mothers are under nutrition until they're weaning. Mm-hmm, until. So I think that the postnatal period, they are under nutrition and the breast fed also is different. Okay, yeah, yeah. Yeah, I would have to, yeah, look into that. Okay, well, thank you. Thanks, Tiffany. Thank you. Thank you. So next up, we have Yanlin He from Pennington Biomedical Research Center. Hey, everyone. My name is Yanlin He. I'm very glad to present our unpublished work here. Thanks organizer to give us this opportunity. And first of all, I want to use this title page to summarize that really the hunger hormone of aspirin will activate the AGIP neuron final activity to promote feeding. I have nothing to disclose here. And our story starts for this rare disease called neuronal progenitor syndrome. You can obviously see that the most phenotype of this patient is they are very lean with almost zero fat. And basically the two patients we published, they have very low BMI with only half of the daily calorie intake and also daily energy expenditure. That's why this patient can still survive. Early in the year 2016, our collaborator, Dr. Atul Chhabra from Baylor College of Medicine first report the cause of these patients. And so really what happened to this patient is they have a mutation happen in this gene called fibrillium one. And because of this mutation, the patient cannot produce this hunger hormone called aspirin. That's cause they are low appetite and also low body fat. And later on in the year 2019, a Chinese group from Tsinghua University first reported that aspirin can bind to a receptor called olfactory OLFR334 in the liver to promote the glucose production. And unfortunately in our study, we actually did not observe a highly expressed of this OLFR334 receptor in the AGIP neurons. Instead, we observed another receptor called the receptor type tyrosine protein phosphatase delta, which is highly expressed in the AGIP neurons. And later on, we proposed that basically aspirin target to the AGIP neuron through the binding of this PDBRD receptor to activate AGIP neurons and further promote feeding behavior and also the body weight gain. Now, through our previous two publications, we basically demonstrate a model that during the diet-induced obesity in mice, animals will secrete more aspirin in the plasma by doing this, aspirin will stimulate AGIP neurons firing activity and finally promote the feeding behavior and also induce obesity. Although we have two publications, however, there's one more question needs to be understood. That is, what is the mechanism for aspirin to activate the AGIP neurons through this receptor? And because I am electrophysiologist, I'm more interested in identifying an intracellular mechanism of this aspirin. So before I go through our data, I want to quickly remind you about our previous publication when I did in Dr. Yunshu's lab in Baylor College of Medicine. In the year 2016, I discovered that hunger will activate the AGIP neurons through the reduction expression of the small conductance of potassium ion channel, or SK3. What I did is, under fat condition, we euthanized animal and stained the AGIP neurons with anti-SK3 antibody, and we found that most of the AGIP neurons express SK3 under fat condition. In another experiment, we overnight fastened the animal and we euthanized animal, collected the AGIP neurons, stained with SK3. Our data indicated that overnight fasting significantly reduced expression of this SK3 ion channel. And this data is also consistent with the previous publication from Dr. Scholter's Sturluson's lab in e-lab paper. So what is SK channel? It is the type of channel called small conductance calcium activated potassium channel. I'm not going to introduce a very detail about this channel, but just remember, this channel have four different subtypes. And when this channel open, they will trigger the calcium, the potassium, driving from the cytosol to the extracellular, which will induce inhibition of this neuron activity. However, if you do opposite way, for example, inhibit this channel or reduce the expression of this channel in neurons, it's gonna activate those neuron. So, first of all, I use two reporter animals. One is the control animal with TD tomato labeled AGIP neurons. The other is this FBM1-NPS, Nockin mouse model. We also call it S-person deficient mouse model. The beauty of this mouse model is Dr. Atul Chhabra Nockin one of the mutation from the NPSP patient. So it's perfectly mimic the link phenotype from the NPS patient. And with this resource, we quickly do another experiment to directly record the SK current in those AGIP neurons with or without S-person in the secretion. And showing here is a director representative trace of SK current. You will see here under fat conditions, control AGIP neurons display robust SK currents. However, in animals without S-person in the secretion, it has increased SK currents. And more importantly, when we overnight fasting those animals, SK current significantly reduced in the control AGIP neurons, but it does not significantly change in the animals without S-person in the secretion. And because I just mentioned SK channel is very important for the neuron firing activity. So we also compare the neural activity between fed or fasted in both control and S-person deficient animals. And the data showing here is in the control animals, if you overnight fasting animals, AGIP neuron will firing like crazy compared to the fat condition. However, if the animals without S person, they no longer trigger an increase of the fire activity in those HRP neurons. So I believe this data indicated that the hunger will activate HRP neurons through the SK channel. Now, the next, we did a rescue experiment. As during the nighttime, we inject the S person in these S person deficient animals. And because of this S control, we used IDG to inject those animals. After overnight feeding, and then we use that animal to collect the brain samples at fat condition, we observed that in the control condition of those S person deficient mice, they still show robust high SK current. However, if you give the S person back, it's going to significantly reduce the SK current. By doing this, it will increase the fire activity of the HRP neurons. And later on, we did another experiment trying to develop a monoclonal anti-S person neutralized antibody. The antibody will significantly reduce the active S person in the plasma. So by doing this, you're going to reduce the active S person in the control animals. So we get three different animals, different group of animals, and give them either IDG or MAB or S person. And then we use them as animals and compare their SK currents. The data indicated that S person will significantly reduce the SK currents compared to the antibody MAB injection group or the IDG control animals. And by doing this, it will also significantly reduce the increased fire activity compared to the reduce of fire activity by reduce the S person in the animals. And later on, we tried another experiment because early this year, we already reported that the PDBRD receptor is highly expressed in HRP neurons. And HRP can bind into this PDBRD receptor. However, we still don't know if the SK currents changed by S person is directly from this PDBRD receptor. So to test this, we generate a mouse model by using the CRISPR-Cas9 technique. A tool generate these AAV vectors that contain the guide RNA to target the PDBRD receptor. And we mix them with AAV Cas9 vectors or control AAV mCherry vectors. So we mix those virus and inject into the HRP cream mice. By doing this, we're able to generate animal model that selectively knock out PDBRD receptor from the HRP neurons or the control animals. And then we use it as animal and test it if S person can still activate those HRP neurons. And the result showing here is without PDBRD receptor expressed in the HRP neurons, S person no longer change the SK currents. Or they can also not change the fine reactivity of those HRP neurons. So this data argues that S person reduced SK currents through this PDBRD receptor. Now, I think the next big question is, can we genetically modify the SK3? So as I mentioned previously, SK3 is one of the subunit of SK channel. And this is only the one channel that highly expressed in HRP neurons. So to do this, we get a mouse string from Dr. Joe Eckers lab in Utis, Wisconsin. They generated a mouse model called HRP CREAR. We cross the HRP CREAR with SK3 LuxLux animals. Upon the animals grows to eight weeks age, we give them tamoxifen. This will specifically delete SK3 in adult animals. So once we have these SK3 selectively knocked out from HRP neural animal models, we euthanize animal and did another quick experiment by incubating those brain slides with control GPO S person. And our data shows that within the control animals, S person will significantly reduce the SK current and also increase the fire activity. However, in the HRP neurons without SK3, S person no longer change the SK current. And also their fire activity are no longer changed by S person incubation. So this data will argue that S person really is talking SK3 to activate HRP neurons fire activity. And last experiment we did is we have those animals validated that they are separately SK3. Then we get those animals repeat experiment by giving the animals with IgG or antibody to target S person. This was significantly reduced active S person in those animals by injecting the MAP. So the data is showing here is in the control animals, if we give them MAP, they're going to reduce S person. And this reduction of S person will significantly reduce the overnight food intake. However, in those SK3 knockout animals, although they have intake high amount of food intake, however, there are no significant changes between the control IgG injection group or the MAP injection group. So this data really argue that S person failed to promote the food intake if we knock out SK3 from the HRP neurons. And lastly, I want to summarize what I show here is, first of all, hunger will stimulate the release of S person in mice. And S person actually activate the HRP neuron fire activity through this PD-BRD receptor. Third, S person fails to change the SK currents in HRP neurons if we knock down PD-BRD receptor. Lastly, S person failed to activate HRP neurons if we knock out this ion channel called SK3. And with that all, I want to thank all the people contribute to this S person project. Dr. Bingfeng is my post-doctor. She's supposed to present this talk here, but unfortunately, because of some personal reason, she cannot make it. But I also want to thank lab member from Dr. Dingshu's lab and also post-doctor from Dr. Chop's lab. They have a lot of contributions to this S person project. We are still working on some other interesting phenotypes for this S person deficient animals. I also thank my collaborator, Dr. Pengming Xu from UIC, and other significant contributors in this project. I want to thank the funding resource from NIDDK and also from ADA. With that all, I'm happy to take any questions if you have. Thank you. Troy Ripke Rutgers University's great talk So did we're all those male mice Excuse me. Were they all male mice? Oh, yes a good question. Yes, okay It's because SK channels are regulated by estradiol and hypothalamus. I'm just wondering if you Would potentially find differences in that signaling pathway between the sexes. Yeah. Thank you, Troy. I think that's very Very good questions. I would like to answer your questions, but unfortunately I'm no longer an expert in this ester, I mean sex difference Projects. I think my previous mentor will focus on this topic in her future research, but I Mean, yeah, yeah, yeah good. I think we definitely would have tried to compare this general difference We in the future we are gonna use some female mice and repeat those experiments see if there's any like significant difference Thanks Great talk Nicole teeny from Stonehill College I'm curious to know if there's any expression in the aspersen Receptor and palm seed neurons and you have any reason to believe that it could be negative negatively regulating those neurons Yeah, that's good question. I think we did standing for PDBRD receptor in the hypothalamus there Certain expression of PDBRD in palm seed neurons and our first publication in National science it indicated that actually aspersen do not Really target the palm seed neurons, but mostly highly expressed is in the HIV neurons. Thank you Okay, great, thanks very much Next up we have Kristen Lendovich from University of Illinois All right All right, hello everyone, my name is Kristen Lendovich I am a postdoc at the University of Illinois at Chicago and Today, I will be presenting my doctoral thesis project which was intestinal FFA 2 and FFA 3 mediate obesogenic effects in mice on a Western diet So I'd like to start out by introducing free fatty acid receptors 2 and 3 which are G protein coupled receptors Belonging to the free fatty acid receptor family and ligands for FFA 2 and FFA 3 are short-chain fatty acids Which are primarily composed of acetate propionate and butyrate and these are a group of key microbially generated metabolites that act as a major energy source for the body and FFA 2 and FFA 3 are highly similar in terms of protein homology but they do differ in terms of where they're expressed as well as their binding affinities to short-chain fatty acids and their downstream signaling pathways And FFA 2 and FFA 3 are globally involved in metabolic homeostasis and are thought to act via a summation of their tissue specific effects Briefly FFA 2 has metabolic functions within adipocytes as well as modulates inflammation within white blood cells FFA 3 is expressed in the nervous system where it's thought to contribute to the gut-brain communication and Previously our group has characterized FFA 2 and FFA 3 within the pancreatic beta cell where they work in tandem to modulate insulin secretion So FFA 2 and FFA 3 are highly expressed within the intestine however, there is a large body of conflicting data regarding their roles And previously many studies involving intestinal FFA 2 and FFA 3 have relied on global knockout mouse models Which has made it somewhat difficult to study tissue specific effects so here we generated intestine specific knockout mouse models for FFA 2 and FFA 3 and This was accomplished by crossing mice that were floxed for either receptor with a ball characterized intestine specific Cree recombinase driver villain Cree And the result is that Cree recombinase is expressed only in the villain expressing tissues Resulting in in an intestine specific knockout of either FFA 2 and FFA 3 and our strategy Also was to characterize these mice in response to an obesogenic challenge using a high-fat high-sugar Western diet and this was chosen because it does have known effects on circulating short-chain fatty acid levels And is also able to induce metabolic stress so our overall goal for these studies is to characterize the intestine specific roles of FFA 2 and FFA 3 in vivo for the first time and We performed two separate but nearly identical studies using the villain Cree FFA 2 and FFA 3 mouse strains these studies used all male mice fed standard chow until 10 weeks of age and The villain Cree mice and their flocks to litter mate controls were then placed on one of two diets Western diet or a low-fat control diet for a total of 25 weeks and throughout the study We performed a number of experiments probing for changes in three major categories Metabolism intestinal physiology and function and microbiome and short-chain fatty acid alterations and today I will only highlight our key findings Okay, so I'm gonna show the data for the vill FFA 3 mice first our first major observation Was that the Western diet fed vill FFA 3 mice weighed modestly less than their Western diet fed Fox counterparts How is it this This is the pointer Okay Oh Okay. Anyway, sorry Okay, so We found that the Western diet fed Vill FFA 3 mice again we'd modestly less than their flocks counterparts and this trend was maintained throughout the study And looking at the actual percentage of fat mass We found that the Western diet fed vill FFA 3 mice were largely resistant to the development of obesity And they had significantly less fat mass than the Western diet fed flocks group And consistent with this the weights of both subcutaneous and visceral fat pads were significantly smaller We performed H&E staining to assess adipocyte Morphology and we observed drastically reduced adipocyte droplet size as well as significantly less adipocyte hypertrophy Which was clearly increased in the Western diet fed Fox mice Weekly glucose measurements were taken throughout the study and revealed that while there were no major changes in the ad libitum state The Western diet fed vill FFA 3 mice had modestly lower glucose levels when fasted overnight We didn't observe any changes in terms of glucose tolerance as measured during an oral glucose tolerance test however fasting insulin levels and insulin levels taken 15 minutes into an OGTT were modestly lower and More so resembled the levels in the control groups and then finally no changes in insulin tolerance were observed during an insulin tolerance test So we then wondered whether these metabolic changes are influenced by underlying changes in core metabolic functions and Mice were individually housed in metabolic cages after 10 weeks of dietary challenge and we observed no differences in oxygen consumption carbon dioxide production nor respiratory exchange ratio and Similarly, we did not observe any changes in energy expenditure nor cumulative food or water intake We also assessed for changes in intestinal inflammation that could arise in the absence of FFA 3 So in both the ileum and the distal colon of the intestine We observed a significant reduction in histological markers of inflammation in the Western diet fed vill FFA 3 mice Including a reduction in inflammatory cells intestinal crypt destruction and the presence of edema So now switching over to the vill FFA 2 study again The Western diet fed vill FFA 2 mice trended very modestly lighter than the Western diet fed flocks group But interestingly this trend disappeared by the end of the study And similarly while percent fat mass was significantly lower in the Western diet fed vill FFA 2 mice at the earlier time points This effect was lost at the later time points in the study Weights of subcutaneous and visceral fat pads were similar between the two Western diet fed groups However, we did notice a moderate decrease in adipocyte size as well as less adipocyte hypertrophy And the emergence and subsequent loss of the different these differences may indicate a compensatory effect that develops in the absence of intestinal FFA 2 Looking at weekly glucose levels in the Western diet fed vill FFA 2 mice We again observed no difference in the ad-lib glucose levels between the Western diet groups However, in the fasting condition after about 10 weeks, we can see that the flocks mice develop hyperglycemia While the vill FFA 2 group maintained somewhat lower fasting glucose levels Interestingly again, this divergence also loses its significance by the end of the study Glucose tolerance was also assessed midway through the study And while we did not see any differences in glucose levels during the GTT Insulin levels trended lower in the Western diet fed vill FFA 2 mice No differences were observed in insulin tolerance But again insulin levels in both the fasting state and taken 15 minutes into an OGTT at the end of the study Trended lower in the Western diet fed vill FFA 2 mice So taken together these data indicate that the vill FFA 2 mice may have modestly improved glycemic control Metabolic cage data taken 10 weeks into the study revealed that while again, there were no major changes in oxygen consumption Carbon dioxide production, RER or energy expenditure We observed a significant reduction in food intake in the Western diet fed vill FFA 2 group And this difference was most noticeable in the ad-lib glucose levels And this difference was most pronounced during the night when the mice are actively eating This reduction in food intake could be partially driving the protection from Obesity and hyperglycemia observed in the mice during the study And interestingly we did not observe any overt changes in histological markers for intestinal Inflammation both Western diet fed groups developed morphological changes in the intestine And had similar histology scores but one consideration here is that these tissues Were harvested at the end of the study at which time some of the major phenotypic differences Between the Western diet fed vill FFA 2 and floxed mouse groups had already disappeared So to summarize the major conclusions from these studies Our primary finding was that the ablation of either FFA 2 or FFA 3 exclusively in the intestine Results in the partial protection from Western diet induced metabolic dysfunction In the vill and cream mice this was not observed in the control diet groups And this indicates the contribution of FFA 2 and FFA 3 To the development of diet induced obesity and hyperglycemia And additionally despite differences in downstream signaling pathways The loss of intestinal FFA 2 and FFA 3 result in similar but not identical phenotypic changes In the vill FFA 2 mice we observed a significant reduction in food intake And in the vill FFA 3 mice we observed a significant reduction in intestinal inflammation And finally the emergence and subsequent disappearance of phenotypic differences In the Western diet fed vill FFA 2 group indicates the possibility Of a potential compensatory mechanism that develops in the absence of intestinal FFA 2 And this effect was not observed in the FFA 3 study So this characterization provided a great starting point for understanding The intestine specific contribution of FFA 2 and FFA 3 And we plan on using this model to answer some outstanding questions First and foremost we're really interested in exploring how the involvement Of intestinal FFA 2 and FFA 3 mediates secretion of gut peptide hormones Which has been previously studied in global knockout models So we plan on doing in vitro secretion studies using intestinal organoids from these mice We'd also like to do a short-term obesogenic challenge on the vill FFA 2 mice And complete some of these experiments before any compensation takes place as well as assess the potential mechanisms of compensation and Then finally we're interested in a more examining the involvement of ffa3 in mediating the effects of inflammation So with that I'd like to thank the members of the laden lab as well as our collaborators, and I will now take any questions Thanks Hi, this is great talk. This is home Sharon from Indiana University I guess you've already answered my first question with which is about the gut peptide release I think that's a great direction to go looking forward to hear your future studies I have another question so you I guess you used the villain Cree mouse I wonder if you have contemplated the idea of using the inducible villain Cree knockout mice We actually haven't I Haven't had any problems with the with the villain Cree model so far We did do a bunch of model validation, so we haven't really looked at an inducible Knockout model at this point. Thank you A Very nice study, so because you're knocking these receptors out in the intestine I'm wondering have you looked at nutrient absorption through the intestine is you know and you look to like what's left in the fecal pellets That kind of thing yeah, so Those are things that I do want to look at it's a matter of finding people with the right equipment But yes, we are interested in that Very nice talk I love it This is the ending here from Pennington my question is do you know if there is any expression of FFA 2 or FF 3 in the brain and Whether they have functions for the feeding behavior other things For FFA 3 yes definitely There was a recent paper about the role in the notos ganglia on mediating the effects of feeding behaviors Which is kind of interesting because we saw a difference in f2 But not f3 f2 has been associated with changes in feeding behaviors But I don't think it's expressed in the brain. I'm not totally positive on that. I think f3 is more of like the Brain, that's good to know. Thank you Thank you for the great talk it sounds like you had a lot to do during a PhD Since the FFA 2 and FFA 3 are shortening fatty acid transporters Is there any data on whether modifying the diet either with specifically a lot more One of the two sisters so straight differences between the two or even deficiency of one or the other from the diet would cause any of these Phenotypes yes, so that that was that that's actually another direction that we're going in is we've been optimizing a high fiber diet For these mice so that's kind of the next step is to explore What happens when you either give them like a bolus of short chain fatty acids, or you give like chronic feeding of? high fiber diet Hello Bernal from Wash U The question is in regards of microbiome, so you change the structure. Did you change the microbiome? Did you test it and second? What is the absorption of other? free fatty acids medium change free fatty acids is it different so it's a Predominance of one to the other when you cannot sort the long the short change free fatty acids, and if you measure Yes, so we I didn't have time to explain it today But we did do a lot of analysis of the gut microbiome and our results were really interesting because we thought that By knocking out the receptors if we would get alterations in short chain fatty acid levels and Differences between the two groups and that would affect the gut microbiome But what we actually saw was that the gut microbiome changed more so by type of diet than by genotype so now we're wondering if Kind of there's more of an inflammatory Or metabolic effect that is is driving these changes rather than like a short chain fatty acid gut microbiome effect Okay, thanks everyone Next up we have Michaela Stokes from UT Southwestern Okay, and you remembered this is the last Okay, thank you. We had some image problems So I had to upload a couple of times. Thank you guys for sticking around I'm really grateful to the organizers for this opportunity to be able to Share with you guys some of my work I am a graduate student in the Krauss lab at UT Southwestern and the Krauss lab is really interested in Molecular mechanisms that regulate a huge very diverse Group of biological systems, and I'm interested in adipose tissue and as it's already been stated this is really rooted in the alarmingly high rates of Obesity, but more so for me. It's the implications of that excess fat tissue on other biological processes and diseases including Every other biological process that we study in the Krauss lab making me really Interested in trying to understand more about adipose tissue and its regulation and As I'm sure everyone in this room knows When we are taking in more energy than we're expending we have to store it somehow and there's really two options for Storing that the first is to take the mature adipocytes that we already have I Don't think I'm gonna be able to figure out the pointer. Okay, and expand those and this is Not the best choice it leads to a lot of inflammation hypoxia necrosis and Implications on metabolic syndrome the other option is to take precursor cells and differentiate those and this kind of allows these adipocytes to share the load and share the burden of this and This makes me really interested in this process of going from a pre adipocyte to a mature fat storing adipocyte And what are some of the molecular mechanisms that regulate this process? And in the Krauss lab we're really interested in PARP enzymes So PARP's are this large family of enzymes that using NAD as a substrate they can add ADP ribose groups on to target proteins and this can can come in a variety of different flavors, so there's The founding members are these poly ADP ribosome Ribose polymerases and they can add either these long Chains or these branched chains and then there's a subset of this family these mono ADP ribosome transferases that add only a single ADP ribose group on to target proteins and It's really not well understood what mar relation or mono ADP ribosylation Can do in the context of regulating biological processes, so I'm really interested in This this subset of this family and what this single ADP ribose could be potentially doing and We generally think of these marts or mono ADP ribosyl transferases as being localized to the cytosol So generally we think mar relation is being cytosolic and par relation as being nuclear and So everything I'll show today is in the 3d 301 system So these mouse fibroblasts that using this cocktail We can induce differentiation and drive the process to becoming a mature adipocyte And this is really tightly regulated by a number of transcription factors including CBP beta this first wave transcription factor or pioneering transcription factor That then goes and binds to the promoter of PPAR gamma and these second wave transcriptures factors and really drives adipogenesis and our lab has also found that par is Dynamic and tightly regulated in this process and so in an undifferentiated state. We have high levels of par relation and early on After differentiation is induced we see decreases in this and it was actually found that CBP beta itself this pioneering Transcription factor is par related in this inhibits its transcriptional activity and This is a western blot showing that so if we take these 3d 301 cells and look across different times of differentiation We can see high levels of nuclear par relation early on and in an undifferentiated state and pretty quickly this drops off and So I took these same samples and I looked at the cytosolic Marrelation and we can see that there's this really nice mirroring effect where we start with low levels of cytosolic Marrelation and this increases in kind of this mid differentiation time and This was really exciting to us But it wasn't very surprising because NAD this substrate that is used for both of these is compartmentalized throughout this process So in an undifferentiated state we have high levels of NAD in the nucleus and upon differentiation NAD ends up in the cytosol So then I was wondering are these marts or is this mar relation important for adipogenesis Or maybe is it just that that par relation needs to get out of the nucleus and therefore It's kind of just a byproduct of that process And so what I did is I did an si RNA screen where I knocked down each of these marts And then I looked at gene expression and lipid accumulation to ask if there were changes in adipogenesis And if we look at adipo q and fab p4 these adipogenic markers Everything is normalized to a control si RNA, so that's this red dotted line So anything below it is less differentiated anything above it is more We can see that part 7 comes out as a really strong contender in that when we lose part 7 we show decreases in adipogenesis and When we look later on and do oil reto to look at lipid accumulation We can see the same thing and we can actually just see this even by eye if we look at the that top Differentiated control, there's this nice red staining, and then when we go down to the part 7 Knockdowns we can see that this staining is lost and when we extract it off and quantify it We can once again see that the loss of part 7 shows decreases in adipogenesis And we can do this with ODP staining and we validated these with CRISPR knockout cells as well So then to kind of try to get at what is part 7 doing in this process? We just kind of wanted to look at what does it look like throughout this process could that give us any clues as to what? might be happening and so we can see upon differentiation pretty early on the expression levels of part 7 mRNA go up pretty early same thing with the protein levels and Then when we look at the localization because part 7 is actually one of the parts that has been shown in different systems to localize Either to the nucleus or the cytosol. We can see that in an undifferentiated state We see part 7 in the nucleus and upon differentiation. This is Excluded and there's only part 7 in the cytosol and this is at the differentiated point of day 3 So it's when that cytosolic marrelation is at its highest point Making us really wonder is that how part 7 is regulating adipogenesis And so I made these Inducible cell lines where I could overexpress either wild type or catalytic dead part 7 to kind of try to get at this question And here we're looking at Adipogenesis just by way of adipoq where we can see if we overexpress part 7 we see this nice increase But and when we overexpress the catalytic dead mutant we can see the same thing. So telling us that the catalytic activity is not The main role of part 7 throughout this process So then we were kind of trying to get at well if it's not that what could it be and so we decided to do a Rescue experiment using a p-par gamma agonist rosaglutazone. So p-par gamma is in that second wave of transcription factors So when we have part 7 knockdown we can see a decrease in lipid accumulation and when we Treat these with rosaglutazone. We can see that This is rescued and this was a really nice experiment for us for two reasons The first is that these 3 2 3 0 1 cells. It's not a perfect system. They're very finicky So one these cells are capable of differentiating and and then secondly Whatever that roadblock is with the loss of part 7 is upstream of p-par gamma's role in this process and so then we kind of wanted to just look and ask well what is upstream of p-par gamma and The main player is CBP beta and we can see that when we lose part 7 the expression levels of CBP beta are fine And there's nothing wrong there. But when we look at these second wave transcription factors P-par gamma CBP alpha we can see decreases in these with the loss of part 7 So I was curious if part 7 might be playing a role somehow in The activity or the function of CBP beta and its role in this process And so I I peed out part 7 and I asked his CBP beta binds to part 7 and we can see that in fact It does and this is really exciting to us Mostly because we had gone through a huge array of things trying to find something that worked and finally something did But also because it it with all the other data It makes sense that part 7 could be acting as a cofactor in some way With CBP beta and regulating its activity So just to summarize what I've shown you and kind of the other things that I've alluded to that are A part of this system that we know from other people's work in an undifferentiated In an undifferentiated state We have NAD in the nucleus and upon differentiation this switches and we end up with NAD in the cytosol we knew from previous studies that Nuclear par relation levels were really high in an undifferentiated state and today I've shown you that upon differentiation We end up with high levels of cytosolic mar relation I've also shown you that in an undifferentiated state part 7 is localized to the nucleus and upon Undifferentiation we end up with part 7 localized to the cytosol We know from the work of many many others that CBP beta upon adipogenic inducers will bind to the promoter of PPAR gamma and Regulate the expression of these other adipogenic markers And I've shown you today that part 7 binds to CBP beta and that when we lose part 7 We don't see these adipogenic markers Expressed leading us to this Working hypothesis that part 7 is a cofactor for CBP beta in some way regulating its activity And this is really exciting to us to kind of start trying to parse out what exactly that is happening there But there are also many other Groups that looking in completely other biological systems have seen part 7 act in this way as a cofactor to regulate other Transcription factors and other biological processes so with that I would like to Thank the Krauss lab mostly my mentor Lee. He's been fantastic He took me in as a very green grad student and gave me a lot of freedom to develop and drive this project that Didn't always make sense, and I think it took a lot of Patience on his part and so I really appreciate all of that support that I got from him And I'm happy to take any of your questions Hi Nick Webster, San Diego So a great talk and some very interesting findings so a couple of questions come to mind And if you see an interaction between part 7 and and CBP beta have you been able to co-chip them on different promoters? We're trying we're trying right now So we'll see we haven't We we just found that they were binding to each other so that is something we're trying But I can't speak to that and then the fact of the Canada catalytically dead one has the same effect So does that exclude my relation as being important for differentiation um? You know it's hard to say because I think that there's definitely the possibility that This if we really wanted to look at the story of Moral ation only and this was a very me being a young Scientist thing the screen probably should have just been for moral ation right and looking at the decrease in that So I don't think that part 7 catalytic activity is important I've shown that through an inhibitor and Mutants, but I do think that there's the possibility of a different one of those marts having a role with Marla tion and that being vital in some way Hey, Michaela good talk well done Craig dog from the UK I'm guessing the answer is no because you haven't mentioned it, but when you Mess around with levels of power pop seven do the other Mara late and pops change Hmm You know nothing stood out when I was originally doing my screen and knocking them down I had looked at all of the different marts when I knocked all of them down Nothing came up, but actually so if we were to look at like RNA-seq data and look at all of the different marts part 7 goes up after differentiation all the other ones go down So I think that it and this is something we've had a hard time with in this project in general if part 7 is acting Early enough how do we distinguish a director and indirect role right is it? Just because we're not at that stage of differentiation because we blocked it early on or is it because there actually is some direct interaction there So I don't know that we we would see anything just because the levels of the other ones do go down, but also It would just be hard to tease out what that mechanism would be Maybe I have a question just to follow up on the Marla tion it goes up in the site is all during differentiation So I'm wondering when you knock down Part 7. What's what's the fractional contribution of part 7 to that correlation? Did you look at correlation in this site as well with part 7 knockdown? So I did and it and you see it you see it Gone, but this gets back to the if I was knocking it down at an early time point am I comparing a day? One or an eight hour to a day three right of the control So then I ended up doing a part 7 specific inhibitor Just like overnight from day 2 to day 3, and we don't see decreases So I don't think that that is all part 7, and there's many others that it could be and then another question Part 7 needs to bind CPP beta, but it's trafficking to the site as well So do you know first of all how it gets to the site as well? And if you mutate that and trap it in the nucleus what happens? So we've done so part 7 has a couple of different domains It has a zinc finger domain and a WWE domain, which means it can bind to par So that's one thing we've kind of thought about is is part 7 binding to CBP beta through CBP betas correlation And I do know that with the zinc finger in the WWE mutants Part 7 localizes to the site assault, but I haven't really tried to like add on Like a Some kind of other import signal and keep it there outside of those mutations that could be its functional role Okay, thanks, thanks very much. Thank you Is run in the audience Okay, I guess he didn't show up so you guys are off early. That's the end of the session today. Thanks very much. Thanks

aspirin

AGIP neurons

feeding behavior

obesity

neuronal progenitosis syndrome

PTPRD receptor

SK3 channels

mouse experiments

rescue experiment

PARP7

CBP beta

adipose tissue