So, good morning everyone. I'm gonna be presenting my work, which is entitled Proper One is Not Essential for Pituitary Cells Terminal Differentiation. First of all, I'd like to thank the organizing committee for letting us present our work here. I have nothing to disclose. So, the pituitary is known as the master gland of the body. It's mainly composed by two parts, which is the neurohypothesis, which is located at the posterior part, and the adenohypothesis in the anterior part. So, the adenohypothesis is responsible for producing six hormone types, which are acting in growth and metabolism, reproduction, and also other body functions. So, in order for the pituitary cells to be, let me see just if I have a pointer. So, in order for the pituitary cells to be differentiated, we need a time and also a space expression of several transcription factors and signaling molecules. Any changes or pathogenic variants in pituitary transcription factors can lead to pituitary hormone deficiency, which is known as hypopituitarism. And among of the pituitary transcription factors, proper one is one of the most important ones, just because it's the main cause of combined hormone deficiencies in the pituitary in congenital cases and also familiar cases. So, the proper one gene is very important for the pituitary, and it's known to be, oops, sorry, it's known to be involved in pituitary cells' specification and also cell migration, leading to dimorphology and also hypoplasia in adult mice, pituitaries. An animal with a proper one mutation, which is known as the Ames mice, is described as GH deficiency, GH deficient, TSH, and also prolactin deficient with reduced levels of LH and FSH. And it's also known in some recent studies in the aging field and also in testing that some male homozygous for the proper one gene can mate with females, heterozygous mice or wild-type mice, and can give rise to colonies and viable colonies in heterozygous mate fashion. So, our aim for this project was to see what was the pituitary, how was the pituitary differentiated cell pool in a proper one model of mutant mice, so the Ames mice, in a high demand period. So, the high demand period that we chose was the sexual maturation period, which is defined as P30, which is the post-natal 30, until P40, where generally the mice are fully sexually maturated, and the adult control at P60 was used as control. So, we have collected 12 samples for each group, only in male mice. All of the samples, all of the animals had their nasal anal length analyzed and also they were weighted, and we collected blood to measure pituitary hormone levels and also pituitaries to analyze the transcription of some pituitary hormone co-defined genes, and we also did immunofluorescence to correlate with protein expression. So, we saw, so in here we can see in the light blue colonies, which are representing the Ames mutant mice, and the dark blue colonies are representing the controls. So, we can see that Ames mice, they are smaller, as already described and already known, and also lighter than their control age-related pairs. For pituitary hormone co-defined genes, we saw that. So, in here we can see a fold change of the mutant related to the Y type, and we saw that GH was decreased at P30, so in the beginning of the sexual maturation period, and at P60 in adult mice, while at P40 it was with expression levels very similar to the controls. While LH had higher expression at P40 and lower expression at P30, and similar expression levels to the normal mice at P60, and FSH was highly expressed between, even in P30 and P40, and lowly expressed at P60. So, for other hormones, as TSH, the mRNA levels were similar to the Y type mice at P30, and decreased either at P40 or P60, and CGA was decreased at all analyzed periods, while prolactin was increased at P40, and decreased in all of the other two periods. So, in general, we could see that some of the hormones in the pituitary had mRNA levels increased at P40, which corresponds to the end of sexual maturation period. So, we also wanted to correlate this data with the serum levels of the hormones, and the bloodstream, so we saw that for LH, we didn't see a change between the mutant mice and the Y type mice, either at P30 or P40. For FSH, at P30, we had a decrease in serum levels, while at P40, we had a similar level to the Y type. For GH, was the same, so reduced levels at P30, and normal levels at P40, and for TSH, we didn't see a change in either of the periods, and prolactin was decreased for both of them. So, in general, we could see that at P60, none of the hormones were similar to the Y type, so they were all reduced, while at P30, we had LH and TSH with similar levels to the Y type, and at P40, we also had GH and FSH included in this list. So, to confirm the transcriptional data at protein level, we also analyzed LH and FSH immunostaining for PROP1 and also Y type pituitaries, and we could see a few cells that were positive for LH, either at P31 and P40, while at P60, we didn't see any positive cells for LH. While in the mutant mice for FSH, we could see much more cells positive at P30, and higher expression at P40, and at P60, we also had a decreased level of FSH. We also wanted to relate these levels, so the hormone levels, with pituitary stem cells, so we did a SOX2 immunostaining and also mRNA data to see if this correlate with the levels of the hormones, and we saw that in the AIMS mice, we had a higher expression of SOX2 in all of the periods, with higher expression at P31, P40, intermediate expression, and at P60, we had few expression in the lining of the pituitary. While for mRNA data, we saw an increase at P30, decrease at P40, and increase at P60 related to the Y type N. So in general, we could say that the pituitary stem progenitor cell marker SOX2 was decreased mainly at P40 compared to the other periods. We also wanted to see if PROP1 were targeting HEZX1, which is its direct target for lower expression, so it inhibits HEZX1 expression, and we could see that at P30, as expected, we had a higher expression of HEZX1 in the absence of PROP1, while at P40, we had a reduced expression, and at P60, HEZX1 levels were similar to the Y type. So in conclusion, we can say that AMES pituitaries, they can differentiate hormone-producing cells, and in high-demand periods, we can see hormones being secreted in the bloodstream of the AMES animals, and we do believe that we might have other players for cell differentiation and hormone production besides PROP1. So in here, we can see that PROP1, in the absence of PROP1, we still can have some pituitary-differentiated cells, but this doesn't reflect in the phenotype of the mice, as they remain smaller and also lighter than their controls. So finally, I would like to thank all of you for listening this presentation, and also all of my lab members that helped me with this work, and I'm happy to answer any questions. Thank you. If you have any questions, please come to the microphone. Thank you so much for that fantastic presentation. I'm Lori Reisman from the University of Illinois. I'm wondering if you looked at PO1F1 or PIT1 expression because we know that that's necessary to get the hormones and so I'm wondering if you see the same type of pattern and then maybe that is being controlled independently of PROP1 or not. Thank you for your question. So we did not look at PO1F1 expression. We did look at other pituitary transcription factors and they all correlate with the data but that's a very interesting thing that we should do for sure. Thank you. Next presentation, this is Dongyun from UCLA. I have a question about the sample collection. When you collect pituitary for PCR and H-staining, have you collected the intermediate pituitary or only anterior pituitary? So, it's the whole pituitary. I collected all the whole pituitaries and they were analyzed on each pituitary, not in pools. Okay, I see, thank you. Thanks for the presentation. Dan Bernard from McGill. I'm just wondering if your housekeeping gene for your qPCR analysis, was that consistent across all of the ages? Yes, so we did have two housekeeping genes, so we used STH, sorry, ACTB, beta, and also GAPGH, and they were all consistent. Okay, because I find it a little bit surprising. Maybe we'll talk about FSH. You showed it at day 40, that there was an increase in FSH beta, but that wasn't reflected by an increase in the serum levels of FSH, and those two tend to go together. So, do you have an explanation for this discrepancy? Yeah, we also were wondering about it, because in the beginning, so we repeated this data a lot of times to really make sure that FSH was highly expressed, because this was also our question at the beginning. So we intend to do mRNA transcriptome to understand what is going on, because we do believe that, we do know that LH and FSH go together, but it actually reflects in the serum levels at day 40, and also in the immunostaining levels. Okay, thanks. I mean, if I remember correctly, the alpha subunit was low at all ages, so maybe that's limiting in terms of the secretion. Okay, thanks. This will be the last question. Hi, fantastic talk. I love this work. I work with Gwen Childs in Arkansas. Is it known that Prop 1 is upstream of SOX2? Is that known? It's downstream. Well, I know, but it's either some kind of... I have known. You drop Prop 1 and SOX2 drops. Yeah, I know. But I do believe that this drop in SOX2 is related to hormone-producing cells. So we don't have data to show this and to make sure that they are correlated, but we do believe that the stem cell pool is differentiating into... And the differentiated cells sort of negative feedback onto the stem cells? Yeah. Something like that. It's cool, thank you. Thank you. Thank you, Juliana, for that great presentation. Give her a hand. Thank you. So our next speaker is Catherine Bronson from the University of Arkansas for Medical Sciences. Nope. Okay, if you could introduce yourself, that would be great. Okay. Thank you. All right, so I'm Angus McNichol. And... Can we have my slides? Yeah, they need to... It's gonna be a very short talk. No, this is for Catherine Bronson's talk. McNichol. Katie couldn't make it, unfortunately, so I'm having to stand in and give the talk for her, so I apologize for confusing the organizers. I'd like to thank the organizers and the co-chairs for giving us the opportunity to present our work to you today. So the anterior pituitary, as you know by now, is comprised of a number of distinct cell types with unique hormone secretion properties. One of the remarkable features of the pituitary is it can adapt to organismal need by changing the proportion of these cell types to make the right amount of protein on demand. We are interested in trying to understand the molecular mechanisms by which this cellular plasticity occurs. Possible mechanisms include proliferation, so you just make more of the hormone cell type that you need, although I note that the proliferation rate in the adult gland is quite low. Alternatively, you recruit the cell type you need from resident adult stem cells in the pituitary gland, and or you can invoke transdifferentiation, where one cell hormone producing cell type switches cell fate to produce a different hormone. Okay, so we would like to know the mechanisms by which this occurs. One potential mediator is a post-transcriptional regulator called Misashi. Misashi 1 and 2 are sequence-specific RNA-binding proteins which have been shown to control cell fate in other systems. They're highly conserved evolutionarily. They're expressed primarily in stem cells and cancer stem cells, and expressed at very low levels in terminally differentiated cells. Misashi functions to enhance stemness, loosely defined, it promotes stem cell proliferation and opposes stem cell differentiation. With relevance to the pituitary, we have shown recently that Misashi 1 and Misashi 2 are present in all hormone-producing anterior pituitary cell lineages, not just the stem cells, which is somewhat unusual, and we have shown that Misashi can repress translation of a number of terminal differentiation markers of hormone production, including gNRH, PRL, and TSH beta. So we hypothesized that Misashi might fine-tune expression in the pituitary and dampen expression of markers of terminal differentiation, perhaps favoring the ability to undergo transdifferentiation. That's the working hypothesis. So we have shown, as I said, that a number of these pituitary mRNAs are regulated by Misashi. Hitherto, we've been looking at reporter assays in a heterologous system. So what you're looking at here is a gNRH reporter assay. In this assay, you fuse the gNRHR 3'-UTR to a 5'-Luciferase reporter, and in comparison to controls, when you expressed ectopic Misashi, you see a repression in translation of the reporter mRNA. This repression is modest. It's about 15% to 20%, but we believe this is physiologically relevant. It's comparable to how much repression you see when a microRNA binds to a target mRNA. So modest, but we believe it's significant. But again, this is a heterologous system, and we wanted to know this actually happening in vivo. So Angela Odell and Anna Maria in the lab developed a gonadotrope Misashi 1,2-null animal, so specifically deleting Misashi 1 and Misashi 2 in gonadotropes. They then fax-purified those gonadotropes and looked for gNRHR protein levels, which are shown down here in the left panel in blue. So compared to the wild-type animals, animals which are gonadotrope Misashi 1,2-null show increased levels of gNRHR protein at two points during the estrous cycle. There is no significant difference in the mRNA levels, so this increase in protein is post-transcriptional. Similarly, they were able to look at the FSH-beta 3' UTR. Again, this is modestly repressed by Misashi in heterologous assays. And again, in vivo, in purple now, you're seeing an increase in both FSH-beta serum, FSH-beta content in the pituitary, without a significant increase in the FSH-beta mRNA. So this is consistent with Misashi exerting translational repression of both of these, since in the absence of Misashi, you're gonna make more protein, which is actually repressive. So we had been looking at kind of a case-by-case basis of whichever pituitary mRNA grabbed our fancy at the time, but Juchan Lim, a graduate student in the lab, decided to take a more unbiased approach, and he performed Misashi RIP-seq and identified over 1,100 mRNAs which specifically associate with Misashi in the adult pituitary. We then validated a number of these, and I want to just focus on one of them down here, PROP1, which you've just heard about. PROP1 marks a progenitor cell population in the pituitary through which all five hormone lineages are derived. Unlike all the other mRNAs we'd hitherto examined, Joel Banik in the lab found that instead of, I knew I was gonna do that, so instead of repressing translation, as you see, for example, the pi1f1 in the green, you actually see translational activation of the PROP1 mRNA by Misashi. So we don't know how Misashi drives translational activation of the PROP1, and we'd like to know. Misashi, I'll remind you, is an RNA-binding protein, has no intrinsic enzymatic activity, so it is thought to exert translational control by recruiting proteins and having altered protein-protein dynamics. So we wanted to find protein partners of Misashi. To do that, we turned to the Xenopus oocyte model system, which is a truly wonderful system, my favorite, but I digress. In the Xenopus system, the immature oocytes are arrested in G2 phase of the cell cycle and are stimulated by progesterone to re-enter the cell cycle, get to metaphase of meiosis II, at which point they're competent to be fertilized. This process of oocyte maturation requires the translational activation of a select number of dormant maternal mRNAs. And we have shown in previous work that Misashi is absolutely required for both oocyte maturation and the select translational activation of these maternal mRNAs. So in this case here, antisense to Misashi blocks oocyte maturation completely, and it blocks the translational activation of those maternal mRNAs I keep alluding to. So using the system, we figured we could get at the protein partners. So we performed immunoprecipitation with Misashi from either the immature or the mature form of the oocyte, performed mass spectrometry, and in published studies, we identified 29 proteins which were associated with Misashi under conditions where it is directing translational activation. So 29 proteins is rather a lot. And so what Katie Bronson in the lab did, and she would have told you if you'd been here, is she performed an antisense screen to knock down each of those proteins individually and see if any of them affect oocyte maturation, and if any of them affected Misashi protein function. So what you're looking at here is oocyte maturation at two distinct time points, halfway through the maturation process or completely matured, and antisense MOS completely blocks this process. So then Katie screened 29 proteins, and she found six which fulfilled the criteria of delaying or blocking oocyte maturation. And we're going to focus on one of these, the LSM-14 family for the rest of this presentation. So the LSM-14 proteins are involved in a variety of RNA metabolism steps throughout evolution, including mRNA splicing, tRNA processing, and mRNA decay. There are at least 18 of these proteins in eukaryotes. Most of them form hetero-hetomeric complexes. However, LSM14 is somewhat unique in that it's monomeric and functions monomerically, and it comes in two flavors, an A and a B isoform. It turns out it is only the B isoform which is necessary for Xenopus oocyte maturation. The A isoform has no effect when knocked down. So that implies that the 14B is giving a phenotype similar to the Musashi knockdown, so it might be part of the pathway. It associates with Musashi, and to get an idea if it actually impinged on Musashi function, we looked at the translational activation of one of these maternal mRNAs, the Musashi mRNA, the Moss mRNA, excuse me, which is absolutely dependent on Musashi function for translational activation. And what you're looking at down here is the protein-dependent accumulation of the Moss protein in a wild-type oocyte. This is completely blocked in the Musashi antisense, and it is also dramatically attenuated in the LSM14B knockdown. Does this have any relevance to the Prop1 translational activation I told you about before? Here is the heterologous assay showing Prop1 translational activation. If we knock down LSM14B in this system, we efficiently lose the protein, and we no longer see significant activation of the Prop1, implying that it is actually a component of Musashi translational activation in this system. This is a single-cell data to show that LSM14B is expressed in male, shown here, and female mouse pituitaries in all AP hormone-producing cells and in stem cells. So it's in the right place to exert translational control. We see a similar pattern when we reanalyze single-celled seq data from human embryonic pituitary data. So in conclusion, I'd like to say that Musashi has a broad range of endogenous target embryonase in the pituitary. It can repress translation of GnRHR and FSH beta in vivo. Musashi directs the translational activation of Prop1, and we've also characterized several others from our screen. Musashi co-associates with LSM14A family members, but it's only the LSM14B which is needed for Musashi translational activation. And I'd just like to acknowledge the people who have done the work and our funding sources. I'd be happy to take any questions. Thank you. that mediates repression between these two genes and are they different? So the field believes that massage exerts repression in part through the poly-binding protein blocking interaction with 4G and the initiation complexes. We're not entirely sure that's correct. And the short answer to your question is no. But one of the graduate students, Sophia Tomlinson, will be doing mRNA pull-down and then mass spec to address exactly that question, what proteins are associating with a repressed mRNA versus an activated mRNA. Thank you. Dan. Thanks for the talk, really interesting. I guess my question is somewhat related. When you look at the sequence to which Musashi binds, is there a particular sequence? Does that differ between the repressed versus the? Full disclosure, I have a very long list of things that doesn't explain activation versus repression. So we looked at that. So we looked at MBE position in a UTR, number of MBEs in the UTR, length of the UTR, whether the MBEs cluster towards the stop codon, whether it's closer to the polyadenylate hexenucleotide. We have been unable to discern any pattern that discerns an activated from repressed mRNA. Now I'll caveat that with we're working with a small number of mRNAs. So we're trying to do a screen at the moment to get up the number of up and down regulated mRNAs by proteogenomics, be able to get a better statistical analysis. But at this point, if you ask me what I think it is, it could either be the structure of the 3' UTR or it's somehow full. So not all MBEs are being utilized in any of these UTRs. And so there's a thought that you need a stem loop or a bulge in the structure to be able to present correctly. So some of them might not be presenting correctly, some of them might be, and or there's an extra motif we don't know about that some other micro RNA or protein binds and that facilitates with actually forming either the repressor or the inhibitor complex. So that's a great question and I would love to tell the answer but I can't. I guess I'm alone so maybe I'll ask a follow up question just about physiological relevance. We've had a few models where we see large changes in GnRH receptor expression at the mRNA level that we can't really associate with a change in GnRH signaling. You're looking at translation and I'm just wondering whether you've been able to detect physiological consequences of small changes in the protein. So actually Angela has done those experiments. So in the gonadotropin-1,2-null animals, we're seeing an increase, about 40% increase in the GnRHR protein at two of those time points. That leads to a blunted LH surge. So and we see a diminished estradiol levels in those animals and the follicles are, there's fewer follicles. So unfortunately there is no subfertility which is kind of what we're hoping we're gonna see. So it seems that we're modulating the levels but the sufficient residual LH surge to be productive, reproductively. But we are seeing physiological changes. It's just not the phenotype we're hoping for. Yeah. Thank you. Sure. Okay, thank you very much. Thank you. Ren? Hello, everyone. Okay, thanks for coming, and I thank you meeting organizers and co-chairs for giving me the opportunity to share our work with colleagues at this Endocrine Society meeting. The title of my talk is Endocrine Mechanisms of an Orphan G-Protein-Coupled Receptor Regulating Metabolic Homeostasis. I have no conflict of interest to disclaim. First, I would like to thank the people who made the critical contributions to this work. They are Dr. Austin Reilly, who graduated and received his PhD degree last year, and Drs. Xiang Yan and Jason Conley here. Metabolism is titularly regulated by homeostatic mechanisms. When these homeostatic mechanisms go awry, metabolic diseases, including diabetes and obesity, occur. Body weight is regulated by energy homeostasis, which is determined by the balance of food intake and energy expenditure. When total caloric intake exceeds energy expenditure, body weight goes up and vice versa. Excessive food intake and sedentary lifestyles in modern societies have undoubtedly contributed to the increasing prevalence of obesity worldwide. Moreover, obesity is associated with a number of medical conditions, including type 2 diabetes and cardiovascular disease. So like body weight, glucose metabolism is also regulated by homeostatic mechanisms. Defective glucose metabolism leads to hyperglycemia or hypoglycemia, and both are detrimental. Glucose homeostasis is maintained by the coordinated actions of multiple organs. These include pancreas, GI tract, brain, liver, muscle, and fat. And so insulin resistance and beta cell dysfunction contribute to impaired glucose homeostasis, which manifest as defective post-prandial glucose disposal, and insufficient insulin secretion. Recent work have provided details about the neuroendocrine and enteroendocrine mechanisms of metabolic regulation. First, there is a complex neurocircuitry within the hypothalamic nuclei. Second, metabolic hormones produced by the peripheral tissues act on the hypothalamic nuclei to regulate feeding and glucose metabolism. These hormones include leptin, insulin, ghrelin, GLP-1, PYY, CCK, et cetera. They are produced by the adipose tissue and the GI tract. Together, they relay the nutrients and adiposity signals from the periphery to the central nervous system. So studies have reviewed critical, studies have reviewed the chemical and the molecular identities of critical hypothalamic neuronal populations that regulate energy homeostasis. For example, there are two key hypothalamic neuron populations, namely AGRP neurons and POMC neurons here. So the current research focus is to investigate the neuroendocrine and enteroendocrine mechanisms of G-protein-coupled receptor signaling for maintaining glucose and food intake. So here is a quick outline of my talk here. So first, I'm going to tell you how our work started, and then I'm going to give you an update about our work in neuroendocrine function of GPR17. And if time permits, I'm going to share my thoughts about the future directions of our work. So we started with investigating AGRP neurons in the hypothalamic acuity nucleus. These neurons express both insulin and leptin receptors, and they regulate metabolism by responding to insulin and leptin, as well as other hormones. AGRP neurons produce orexigenic neuropeptide AGRP and NPY. In contrast, POMC neurons produce anorexigenic neuropeptide alpha-MSH. AGRP neurons functionally antagonize POMC neurons through AGRP, which is a natural antagonist for alpha-MSH. And in the end, AGRP neuron activation will increase feeding and reduce energy expenditure. So when I was a fellow, I was working on transcription factor FOXO1, and it's both insulin and leptin signaling pathway impinged on FOXO1. So I generated a FOXO1 knockout on animals, specifically in AGRP neuron. And then I characterized the metabolic phenotype of these animals. Long story short, these animals have favorable metabolic phenotypes, including reduced feeding, reduced fat mass, and increased peripheral metabolic flexibility, and increased central sensitivity to hormones and nutrients. But FOXO1 is a transcription factor, and I want to characterize what's mediating its function in AGRP neurons. So I performed transcriptional profiling by sorting out AGRP neurons from wild type and knockout mice. And here I show that GPR17 expression is remarkably suppressed in the FOXO1 deficient AGRP neurons. And a couple years later, a European group had a similar finding and reported their studies. So now I've shown you, we think GPR17 could be a potential target for metabolic regulation. But to further test it, we generated animal models. Okay, we started with AGRP neuron-specific GPR17 knockout mice, and indeed, we are delighted to find that they largely recapitulated the FOXO1 deficient animals. As you can see, AGRP neuron-specific GPR17 knockout mice have less fat mass compared with control animals, and they eat less. As shown here in the black bar, they eat less than their litimate controls. During dark phase, that's when mice were most actively engaged in feeding. I further challenged the animals with a fasting refeeding paradigm. During fasting, GPR17 deficient animals have less locomotor activity, indicating less food foraging behavior, while after refeeding, these animals eat significantly less, indicating less rebound hyperphagia. So altogether, AGRP neuron-specific GPR17 knockout mice seems to have increased satiety. And I also characterized the AGRP neuron and POMC neuron activation during fasting and refeeding. Specifically, during fasting, AGRP neuron-specific GPR17 knockout animals have reduced AGRP neuron activation, and while after refeeding, they have more POMC neuron activation. This phenotype is consistent with the feeding behavior and energy phenotype we have observed in these knockout animals. And my student, Austin Riley, did a follow-up study and then lead the investigation of POMC-specific GPR17 conditional knockout mice. He sorted out the POMC neurons from wild-type and knockout mice, and indeed, his cellular fractions are enriched with POMC transcript, yet the knockout POMC neurons are critically deficient of GPR17 expression, shown in panel B and panel C. After confirming this, Austin did a comprehensive metabolic analysis of these animals, and the most remarkable finding was that these animals have a sexual dimorphic phenotype. And especially when you put them on high-fat diet, the female knockout animals are better protected from the metabolic derangements caused by the high-fat diet. For example, here, female on high-fat diet have less body weight and less adiposity, and they have less adiposity gain, as well. So what could be the underlying cause of this? We collaborated with Sharon Wurlow at Columbia University and performed a blind study, by which we dissected the hypothalami from the wild-type and the knockout animals, and measured the protein neuropeptide level of alpha-MSH, beta-endorphin, and POMC, as you can see here, especially in the females. We observed increased POMC processing in the knockout animals. Austin also performed a blind study, and found that when we knock out GPR17 in POMC neurons, there's increased action potentials in these neurons. So we have published this result. If you have questions, I'll be happy to answer. And I'm going to give you our most recent finding about the leptin sensitivity data. This is not published yet, but I welcome any question. Due to the time limit, I'm just going to give you a highlight of a couple of animal models. For example, we have AGRP neuron-specific GPR17 knockout mice. We found they have less circulating leptin, indicating they may have increased leptin sensitivity. And indeed, when we use phospho-STAT3 as a surrogate for leptin sensitivity, we found that these animals have increased STAT3 phosphorylation in the medial basal hypothalamus. Moreover, we expanded the study to look at GPR17 activity in leptin receptor-expressing neurons. So we created the leptin receptor-expressing neuron-specific GPR17 conditional knockout mice, and we found that these mice are better protected from high-fat diet-induced excessive caloric intake, shown in panel A here. When we switch the mice from child diet to high-fat diet, the knockout mice have less excessive caloric intake compared with controls, and also they have less adiposity gain as a result. We also measured their whole-body knockouts to exogenous leptin injection, and as shown here, the knockout animals have more rapid decrease of food intake and also increased STAT3 phosphorylation in the medial basal hypothalamus. So I am going to stop here and thank all the lab members, collaborators, and Endocrine Society. And this is a QR code. Thank you. Great talk. This is Yanning He from Pennington Biomedical Research Center. I saw you have a lot of good phenotypes of a knockout GPR17 from AGLP and palmitoyl neurons. What about energy expenditure? Have you tried to look at that? Thanks. Okay, thank you, Yanling. That's an excellent question. Thank you for bringing it up. Yes, we measured energy expenditure. Total energy expenditure in the AGLP-specific knockout mice was comparable with the wild-type control animals, yet they have less locomotor activity. So if you average the energy expenditure per locomotor activity count, they have higher energy expenditure. So in other words, they are less energy efficient. So, thank you. Can I just quickly ask if there's anything known about the coupling of GPR17 or any efforts on de-orphanization? That's a fantastic question. That's one of our future directions because we are interested in the therapeutic perspective of this GPCR. So we are looking at its molecular signaling properties and we also identified human genetic variants specifically for GPR17. And we quantified these non-synonymous genetic variants and their impact on downstream cyclic AMP, calcium and beta arrest and recruitment in our GPC paper published earlier this year. Thank you. I was delighted to see that you saw the effect with high-fat diet, not just regular chow. As a matter of fact, I think it was even enhanced with high-fat diet when you knocked out the GPR17. This is related question, are there any pharmaceuticals existing that target that pathway? Oh, this is a great question. So this is another future direction. So we think this is an orphan G-protein coupled receptor, but there are chemical compounds that can activate the GPCR signaling, but we don't know whether they are endogenous ligand or not. And there's antagonist, not specific for GPR, this GPCR, but has been used for the signaling studies. Thank you. Thank you, Shanti. Thank you. So our next speaker is Sanjay Jumani from NIH. Okay, hello everybody, my name is Sanjay Jumani. I am a first year fellow in endocrinology, I'm doing combined adult and pediatric endocrinology. This is going to be a little bit of a departure from the talks we've been hearing. Our basic science colleagues, you guys are doing some good work. This is a case report where we see an association between signal transducer and activation of transcription in the resonance STAT mutations and galacteria. So the objectives of this talk are first to recognize that mutations in STAT genes may lead to galacteria, which has not yet been described, and to understand that intracellular signaling using the STAT pathway may be involved in the production of human milk, and this may be a potential target for therapies for those seeking assistance with lactation. So just to give you some background, prolactin is responsible for the development of mammary glands and milk production, and JAK-STAT signaling is crucial for intracellular signaling of many metabolically relevant cytokines and hormones. The ones we most commonly associate are leptin, growth hormone, erythropoietin, thyrotropin has more recently been associated, and of course prolactin for our talk. And hyper-IgE syndrome, otherwise known as Jobe syndrome, is a well-described immunodeficiency that's associated with several mutations, including STAT3 loss-of-function mutations that account for 70% of Jobe syndrome cases, as well as STAT1 gain-of-function mutations. Intracellular STAT signaling leads to production of bioactive cytokines using cellular immunity, so we're talking IL-6, IL-10, interferon-gamma, and the development of specific B cells, which can account for some of the hyper-IgE seen with this syndrome, and is what causes the immunodeficiency. STAT1 and STAT3 express transcription factors that also mediate prolactin's intracellular signaling, and we'll get into that a little bit later. An association between STAT gene and protein function and galacteria has not yet been identified, or at least reported in the literature. So here we describe three patients with STAT mutations that have had galacteria. So our first case is a 27-year-old woman with a hyper-IgE syndrome due to STAT3 deletion mutation, the valine 463 deletion. It was associated with recurrent infections, as is the case with Jobe syndrome. She had a thin white bilateral discharge with breast manipulation beginning in adolescence. There was no concern for breast pain or abscess. Her prolactin was slightly elevated to 27.9, which is just above reference of 25, and her anterior pituitary panel was otherwise normal. She is on no medications that are associated with hyperprolactinemia. Here's a picture of her pituitary, which showed a 4x5 millimeter microadenoma not causing any mass effect. So you can see here, oops, you can see here, right here, it's fairly obvious. Our next case is a 32-year-old woman with Jobe syndrome due to heterogeneous loss-of-function STAT3 mutation, similar to the first one. It was a deletion. This is a loss-of-function. It is R382q substitution. Also likewise associated with recurrent infections and papillary thyroid cancer, which we're not going to get into now, but it's also associated with Jobe syndrome and STAT3 mutations. She had unilateral chronic thin white nipple discharge with light touch of her right breast for quote-unquote as long as she could remember. She had a history of breast abscess, but no abscess was noted at the time of our exam. Her prolactin was right in the middle of reference range, so normal, and her other anterior pituitary hormones were also normal. Her MRI pituitary was also normal. And then our last case is a 22-year-old woman with a STAT1 activating mutation due to E235A substitution associated with recurrent infections, autoimmune hypothyroidism, type 1 diabetes, and hypogonadotropic hypogonadism. She reported bilateral galacteria starting at age 18. Of note, STAT1 has been associated with other hormone, intracellular signaling, which can explain some of these autoimmune endocrinopathies and other endocrinopathies that she has. She had a white milky discharge that could be expressed with light manipulation, but wasn't experiencing discharge on a day-to-day basis. Her prolactin also was right within the reference range, and she had serial dilution done in her labs, which weren't done on the other two, but those ones were also consistently within the reference range. Her anterior pituitary evaluation was normal, except for elevated TSH, which is consistent with autoimmune hypothyroidism, which was treated. Here's her pituitary MRI, which reveals a diffusely enlarged pituitary without a focal adenoma. It's about 12 millimeters, 1.2 centimeters in the cradiocaudal dimension, and there was no focal hypoenhancement. So JAK-STAT, the pathway is involved in prolactin signaling. As you can see here, STAT1, STAT3 are both associated. Their mechanisms of action are not fully understood, and we don't necessarily know what their downstream signaling includes, but we do know that STAT knockouts do cause defects in prolactin signaling. An analysis done by Sharp et al. looked at human milk cells at various stages of life, particularly in the three phases of pregnancy, as well as postpartum, during lactation, and looked at human milk cells. They found, by doing RNA transcriptome analysis, that STAT3 was upregulated and STAT1 was downregulated during lactation. So these three cases that we had here provide clinical evidence for mechanization between STAT signaling and galacturia. It suggests that STAT3 and STAT1 function in opposite directions, which is consistent with Sharp et al., to regulate lactation. Increased STAT1, decreased STAT3, in our cases, are associated with inducible galacturia. So our areas of further interest and research, first is to elucidate the mechanisms by which seroprolactin feedback loops are involved, as only one of our three cases had an elevated prolactin. It's not clear what the chicken and egg here is, but we hope to identify that soon. We will identify processes by which structural pituitary findings are found. Two of our cases did have findings on pituitary MRI, however, they could be implicated in other hormone signaling, including growth hormone and, like I mentioned, autoimmune hypothyroidism in our STAT1 case. We also have future plans to partner with our immunology colleagues, who are studying Job's syndrome, to identify other possible patients who may have this clinical finding and obtain further lab work and obtain cells from them. Beyond the understanding of specific intracellular signaling pathways, which is our next step leading to galacturia, these observations may be utilized for developing therapies. First to assist those with impaired lactation or to induce lactation for families in whom physiologic lactation is not possible, so adoptive parents, transgender parents. There are two commercially available drugs that affect STAT signaling that could be novel non-dopaminergic galactagogues. The first one is nitroferoxazide, which is a cell-permeable, orally-available antidiarrheal, which suppresses the gene activator STAT1, STAT3, and STAT5, so all three are implied. Fludarabine is another one. It's currently used for treatment of CLL. It's a STAT1-selective inhibitor with specific depletion of STAT1, but not the other STATs. At this point, it causes a lot of systemic toxicity, and I don't believe that any reports of galacturia have been made. However, we can hypothesize that a more lipophilic version of fludarabine could be used for this patient population. These are my references, and I want to thank you for taking the time to listen to my presentation. Thank you. Thank you, Sanjay. Questions? Thank you very much for these studies. I was wondering, in case three, for example, you mentioned a hypogonadotropic hypogonadism. Could you hypothesize that this hypogonadism was due to an activation of the proactin signaling pathway in the gene-RH neurons? Yeah, I think that's the logic. STAT1 is more ubiquitously expressed within both the pituitary as well as downstream pathways for other hormones. So I think, yes, in the FSH pathway, it has been implicated. It's not as strong evidence as leptin or thyrotropin yet, but it has been implicated so far. So I think that's the logic. And if I may, second question on case one, it was an inactivating mutation? It was a deletion mutation, yes. So how do you reconcile this point with the phenotype? So the first two cases were both inactivating mutations, and they were both in STAT3. And the last case was an activating mutation of STAT1. And there was a paper done looking at mammary cells in vitro that showed that STAT1 and STAT3 act in opposite directions. So I think that's, you know, we don't have a mechanism as of yet, a pathway as of yet, but I think that it probably goes with our knowledge of how STAT1 and STAT3 interact with each other in mammary cells. Thank you. Yes. Next talk, I have a follow-up question on case one. You mentioned this patient has a germline STAT3 mutation. It's more like a loss function mutation. Can you, like an image, if we apply STAT inhibitor for treatment, what kind of STAT inhibitor will choose for this kind of germline mutation? Do you think this inhibitor will be a choice for treatment? I think an inhibitor for a deletion is a little bit of a stretch at this point, you know, because we know that in normal lactating cells, STAT3 is upregulated. So an inhibitor probably wouldn't be as effective, but a STAT1 inhibitor could be effective because it might be in the same pathway. So that's a good question. I don't know that we have, we would have to find a way to induce STAT3 production. Thank you for your question though. Can I ask you a quick question? Sure. I just think it's really interesting that you have this mutation, the inactivating mutation, that it seems to be, I don't know, I would have thought STATs are so important that there would be other things going on with this individual. So is there any kind of indication at how it might be very, kind of, sort of inactivating maybe more for the prolactin receptor perhaps to other cytokine receptors? Is there any kind of information on that? Well, so all of these patients, you know, I focused on their endocrinopathies, but they actually do have quite severe immunodeficiencies. That's actually how they presented to the NIH, and this happened to be one of the symptoms they had. So I don't know. I think that these mutations have variable penetrance, and so obviously not everyone with STAT mutations has a lacteria or any of the endocrinopathies associated. So I think there is variability there. Okay. Thank you. Thank you. Okay. Thank you all. Thank you very much. Thank you, Sanja. I would like to thank the Endocrinal Society for the opportunity to present our work. I don't have conflicting views regarding this presentation. The Delica-1, also known as a pre-adiposity factor 1, is a component of the NOTCH signaling pathway. It's located along the arm of chromosome 14, and it's a preteen gene, means only one copy of the gene is expressed in the case departed in a lab. The locus where the gene is located is associated with Temple syndrome. The canonical sequence of Delica-1 genes, it's compounded by five axons. The full-length proteins are composed of six epidermal group factors like tunnel repeats that constitute the major part of extracellular domain. Also has IL-17 cleavage sites in the just the membrane region, which allows the release of soluble form of Delica-1. Also has a transmembrane and a short intracellular domain. Several pathogenic variants in Delica-1 genes has been identified in patients with central precocious spirit. All variants described are small deletions linked to a truncate protein, and all of them are located on extracellular domain. Considering what was said, our aim was to investigate genetic alteration of Delica-1 in a French cohort of children with idiopathic central precocious spirit. The cohort studied was composed of 122 patients of French patients, 98 girls and 23 boys of 75 sporadic and 46 familial case. As inclusion criteria, we considered onset of secondary sexual characters before age 18 girls and 19 boys, bone age accelerated growth velocity, bone age advancement, early age concentrations at baseline and after stimulation at pubertal levels, and MRI without abnormalities. As method, the DNA of selected patients was extracted from peripheral blood. The entire code region of Delica-1 was amplified by PCR, following by sequence by Sanger method. In silica analysis were performed by computational programs, and the database analyzed the classification of the allelic variants. As the first results, we found one allelic variant, which is a switch of acetosine to adenine at position 372. It leads to change of cysteine at 124 position to a stop codon. Segregation analysis showed its in-heteroform the symptomatic father. The patient was first evaluated at 5.5 years, with the Lark-34, advanced bone age, and early age at pubertal levels. MRIs are normal and the patients were treated. The allelic variant is located at axon 4 and results in the premature stop codon, leading to a shortened polypeptide. It is a rare variant with a very low allelic frequency and classified as like pathogen according to SMG critters. It is not reported at ClinVar and not associated to CPP. As second results, we found another allelic variant, which is a switch of thymine to aguanine in the second nucleotide. This leads to a change of the first methionine to adenine. The patient was first evaluated at 7 years old, with history of the Lark of 6 years. She has bone age advanced, early age at pubertal levels, normal MRI, and she was treated. She has one sister with early menarche and half sister with history of premature sexual development. The allelic variant is located at axon 1 and affected initiation codon. It's a very rare variant absent on that base. It's classified as likely pathogenic according to SMG critters and not associated to CPP and not reported at ClinVar. In conclusion, we identified two new rare pathogenic variants in DLQ1 gene in two unrelated French patients with CPP. Both allelic variants were predicted to be deleterious in silicoanalysis. This top gain was inherited from the symptomatic father, suggesting a familial case without previous history of early development in their relatives. I would like to thank our CPP Brazilian group, especially to Ana Cláudia for all the support she always giving, example of woman research and mother. I would like to thank Dr. Raja Brown for the lovely collaboration, and I'll offer you for attention. Hi, I'm Rana Carol from Boston. Did the patients that you had variants in have any metabolic phenotype? Did you look at that? We still not have this data about the metabolic of the patients, but we know they are at least now they still, we know they aren't fat yet, but we're still running the metabolic specifically to know if they have the metabolic alteration as we expect by the DLQ1. They have this associated. Quick question, have you checked the pituitary hormone in this patient population? Sorry? Have you checked any pituitary related hormones in this population, the patient population? I didn't understand that question, sorry. She asked if you checked for pituitary hormones, hormones in the population. No, we didn't. Sorry. Thanks for the presentation. I may have missed this, or maybe I just don't know this literature very well, but I missed the link between DLK1 and central precocious puberty, so is DLK1 somehow regulating cispeptin or GnRH secretion? The way DLK1 act and the puberty, we still don't know. We have some cases of central precocious puberty and DLK1 loss of function mutations, but we know that DLK1 is part of not signaling, and not signaling is important for neuroendocrine development and also adipose differentiation. But exactly how it works, like we know how mRN3 act on the puberty development, we still don't know. But we know he has some part of this structure. Okay, and then if one were to look at, say, a rodent model, or maybe even in human, just an expression, is DLK1, where would you find it? Is it ubiquitously expressed? The DLK1 expression is high in the prenatal, especially in animals, it has been shown that it's high. And the pubertal time is decay, keeping only in some specific tissues, like the endocrine tissues. But we still have to understand how exactly it works. Okay, thank you. And Luciana, can I just ask if DLK1 knockouts have been done, and do they have a similar phenotype as what you've identified in patients? No, they are already done, this knockout. They don't have alteration of the weight, but they have alteration of the development of the pubertal time. Thank you. Yes, so that concludes this session, so I'd just like to thank all the speakers and to all of you for your great questions. Thank you.

neuronal STAT3

aging

neuroendocrine regulation

proper one

pituitary cell differentiation

GPR17 mutations

metabolic homeostasis

STAT mutations

galactorea

endocrine disorders