Okay, well, I'll tell you what, I'll get started with an introduction for Dr. Guo, and you'll hear me be a little bit informal. I'm going to call her Grace, and I apologize, but I will tell you kind of why. So it's my pleasure to introduce Dr. Grace Guo, who's going to be telling us a little bit about the biological effects of bile acids, potential activation of cell signaling pathways, cytotoxicity, what have you. I have known Grace since 2007, where we were among the faculty that kind of met up at the University of Kansas Medical Center. And I will start, normally the introduction would be to tell you about all of the degrees that someone has, and I'll try to cover that. But I want to make the point that Grace is just an outstanding human, and is one who has just poured herself into her science and her career and in her people, and that's evident on her CV. Her training had her in the laboratories of Frank Gonzalez, training with Kirk Clawson as well, and she's risen through the ranks. She's now a professor out at Rutgers, where she's been for, oh gosh, yeah, no, like 12, 15 years or something like that, I forget how long. But having trained with all of these people that gave her the, or helped her earn the skill set that she has to be successful, her name is now on that list, right? So now, when people who have worked for Grace go elsewhere, they're like, well, they trained with Grace. Well, oh, that's why they're outstanding. Her research program has focused on a number of topics, including nuclear receptors, liver diseases of all different flavors, with an emphasis on bile acids. She sold numerous NIH grants, I didn't even count the papers. She has all of the anticipated publications that would come with such a storied career. And not only is it about numbers, it's about impact. And not just about the impact factor of the journal. The papers are highly cited because in many cases, they have caused the field to take a right or a left turn at a fork. Her science has very much driven where the field has gone. And I will let her start her talk, but I want to make one additional point on her CV. She knows exactly where all of the trainees from her lab are, and she catalogs most of their awards before her own on her CV, which I think speaks to, you know, she very much knows the mission of an academic scientist, and I'm very much looking forward to her talk. Welcome, Dr. Guo. Thank you so much, Jim, Liddy, for inviting me. Thank you for that generous and kindest introduction. I am really honored. All right, so today I'm going to talk about the bioassays-mediated gut-liver crosstalk. So for today's talk, we're going to cover bioassays, especially species difference between humans and the mice. And then we're going to talk about FXR as a target in mesh development and how this target has been not successful so far. And then another factor we're going to really emphasize is FGF1519, which is a major FXR target in the gut. And then to point out what are the future challenges that can better use this pathway or some of the genes as drug targets. So we know bioassays are synthesized mainly in the liver by two pathways. The classical pathway and alternative pathway. Classical pathway start with CYP7A1, followed by CYP8B1. An alternative pathway started by CYP27A1, followed by CYP7B1. And then these are considered primary bioassays, including colic acid, chenodeoxycolic acid, and in mice, the muricolic acid. Once they are conjugated, they will be exported into the bilinary tract and then transported to intestine to facilitate lipids or lipid-soluble vitamin absorption. Once lipids are absorbed, then the bioassays will be transported back into the liver through the atherohepatic circulation. For the last 30 years, we know bioassays are not just detergent. They actually imported endocrine molecules to activate nuclear receptors, as well as membrane receptors. So we all know they activate FXR to regulate bioacid homeostasis. They regulate PXR and then vitamin D receptor to detox toxic bioassays. So we know they also activate the G-protein coupled membrane receptors, including TGR5, which are important in regulating energy homeostasis because activation of TGR5 is important in the regulation of production of GLP1, which we all know is the most significant bodyweight losing drug right now. In addition, bioassays can also activate SVAR-PBR. And then for the last five years, especially sulfated bioassays in humans can activate this G-protein coupled membrane receptor called MRGPRX4. People just call it HX4. This is significant because in the past, we always puzzled about what bioassays are causing this severe itchiness in patients, especially with cholestasis and cirrhosis. And then it looks like right now, at least there are some good evidence showing that sulfated bioassays, by activating this G-protein coupled membrane receptor, could be responsible for bioassay-induced severe itchiness. And then, so as scientists, we all use mice, most of us use mice to study human-related diseases. However, there is a clear specious difference between human and mice for bioassay synthetic pathways. We mentioned early on, in humans, bioassay are deoxycholic, CA, choleic acid, deoxycholic, kinodeoxycholic acid. But in mice, these bioassays are converted by a P450 enzyme called CYP2C17 into muricolic acid. So, the muricolic acids are more hydrophilic than mouse, than CdCA, the human primary bioassays. So therefore, rodent has a more hydrophilic bioassay profile compared to humans that we are more hydrophobic. In addition, once bioassays completed lipid absorption from the gut, we know majority of them will be traveled back into the liver by atherohepatic circulation. However, some percentage, like somewhere from 3% to 5% of bioassays will be converted into secondary bioassays. And we know, though, the secondary bioassays levels are highly associated with human disease development, including liver disease and gut diseases. But in mice, deoxycholic acid can be converted back into a choleic acid by this P450 enzyme CYP2A12. But we humans, again, don't have this enzyme. So therefore, this indicates another species difference between human and mice. Therefore, when we use mouse models to study bioacid or bioacid-related human diseases, we have to put into perspective when we interpret the data. So in addition to just regular biotype mice, over the years, we have developed all sorts of genetically modified mice models, mouse models, to study bioacid homeostasis, including the knockout mice for bioacid synthesis, conjugation, and also germ-free mice so that they don't have the secondary bioacids. And then recently, there's two or three labs discovered, basically made, the humanized bioacid mice. Basically, it's the CYP2C17 and the CYP2A12 double knockout mice. These mice also have problems. Although they now do have a bioacid profile reflecting that of human, for example, they have CdCA rather than muricolic acid, but because the mice probably innately does not really have mechanisms to deal with hydrophobic bioacids, those mice, especially the female mice, are shown quite severe cholestasis since birth. So therefore, we still don't really have a humanized bioacid mouse model yet. Recently, my lab also developed this low bioacid mouse model in the hope that we can use this animal model to study individual bioacid signaling so that we will know more about bioacid biology. And then by using this model, we did discover that, for example, UDCA, which is a therapeutic agent to treat cholestasis, is the erythrodeoxycholic acid, also called a Bayer cholecic acid, is actually an FXR agonist in the gut, rather than people have believed that UDCA is an FXR antagonist in the gut. However, as I'm showing here in the right panel, even using this genetically modified mice, the mouse phenotype does not really mimic human phenotype. A good example is this CYP27A1 knockout mice. In humans, the mutation of CYP27A1 gene leads to a severe disease called CTX. So the patients have early onset of atherosclerosis, as well as other diseases. But in mice, they perfectly fine. And then now we know that because in mice, this pathway actually generates an FXR antagonist rather than an FXR agonist. So therefore, the FXR knockout mice mimic the phenotypes in human CYP27A1 mutation rather than the mouse itself. So after we talk about so much FXR, so let's touch base on what FXR is. FXR was cloned in 1995, and it is a ligand-activated transcription factor highly expressed in the liver, intestine, kidneys, and organs not exposed to high levels of bioassays, such as adrenals. It has been shown as the major regulator of bioassay homeostasis, and the bioassays are discovered to be the innate or endogenous ligands of FXR. Especially chenodioxycholic acid is the most potent bioassay in activating FXR. In addition, we also know that some other bioassays, like muricolic acid, actually works as an antagonist of FXR rather than an agonist. And then, as we mentioned early on, this is because the gene CYP2C70 encodes the enzyme for making muricolic acid in mice, but not in humans. We know FXR is important in regulating bioassay homeostasis. So studies from Steve Cleaver's lab, John Chen's lab, Frank Gonzales' lab, and my lab has collectively discovered that in the liver, FXR can induce CYP, and then downregulate CYP7A1 and CYP8B1. But CYP actually plays a major role in downregulating CYP8B1 rather than CYP7A1. In contrast, bioassays in the intestine can activate FXR, and that leads to production of FGF50 in mice and FGF90 in humans. And then this endocrine fibroblast growth factors can activate FGFR4, which is coupled with beta-calcil in the liver, then to activate MAP kinase pathway to suppress bioassay synthesis. So therefore, the liver is very important in regulating bioassay hydrophobicity, but the intestine is very important in regulating the amount of bioassay synthesized in the liver. FGF50 and FGF90, as we mentioned early on, belongs to the endocrine fibroblast growth factor family. The other two are 21, which now plays an important role in lipid and fatty acid metabolism. And then 23 is important in regulating calcium homeostasis. FGF50 and FGF90 are both highly expressed in idiom, the last part of small intestine. And then they bind specifically to FGFR4, not other FGFR receptors. But at high concentrations, FGF90 can also bind to R1 to a certain degree. They are the most potent target genes of FXR in the small intestine, and then plays an important role in suppressing bioassay synthesis, promoting protein production, and regulate energy homeostasis. There's also human and mouse species difference between FGF50 and FGF90. It's because FGF90 is also expressed in gallbladder and some colon cell lines. And during cholestatic situation, human livers can express FGF90, whereas the mouse liver does not. So therefore, this is another interesting finding that humans have additional protection to downregulate bioassay synthesis, especially during extrahepatic cholestasis. Whereas mice, the situation gets worse because bioassay synthesis actually went up in mice with biodegradation. But FGF90 is interesting in humans is because it's also highly associated with human HCC. Like several GWAS studies discovered, HCC patients also have increased FGF90. Whereas in mice study, mouse studies, FGF50 actually suppress HCC development. So we really do not know what's the underlying mechanism. Is that just association of induced FGF90, or FGF90 actually is a mitogen in HCC development? So one important role of FXR is in regulating liver lipid as well as inflammation. So this is a topic everybody knows for this audience that, especially in the United States, as well as many parts of the world, this metabolic dysfunction associated steatohepatitis, used to called NASH, now called MASH, is a huge medical burden for the medical society, as well as alcoholic-related steatosis. So this actually is a beautiful example of collaborative study between me and Jim. So Jim were junior faculties in the Department of Pharmacology in Kansas. So one day, Jim and I were talking about NASH. This is the time, like 2007, 2008. NASH was just become an interesting topic in the hepatology field. And then I was studying atherosclerosis at the time. And then when Jim mentioned, I remember we have some really fatty livers after we fed the mice with high-fat diet in the APOE and LDL receptor knockout mice. So Jim and I quickly put together a study in three months. And this is the first paper published regarding the role of FXR in NASH development. So the take-home message is, if mice do not have FXR, then they have increased steatosis and fibrosis in the liver, inflammation as well. So indicating FXR could be an important factor mediating NASH development. In addition to NASH, both Frank Gonzales' lab as well as David Moore's lab simultaneously reported that FXR deficiency can cause spontaneous HCC in mice. In my lab, we also reported that in human HCC patients, if the patient not only has decreased FXR expression, but also have decreased FXR function reflected by the decreased expression of FXR target genes. And this is probably due to either KRAS interruption as well as menselation. And this also happens in colon cancer, although mice without FXR do not spontaneously develop colon cancer, but they are more susceptible to colorectal cancer development. So with follow-up studies, we propose that FXR can suppress carcinogenesis by two mechanisms. One mechanism is FXR can induce some of the tumor suppressor genes, such as SHIP, SOX3, e-cadherin, to inhibit cell proliferation. And then another mechanism is by downregulating bioassays. So we know bioassays can increase STAT3, beta-catenin, MAP kinase activation. So this is another pathway that we could reduce cell proliferation and reduce tumor genesis, at least in the liver. Because of all this interesting or beneficial findings that FXR activation is associated with housing the liver, reduce static cell activation and proliferation, and also homeostasis in the gut to regulate bioassays and other lipid homeostasis in the liver, as well as to reduce inflammation, FXR, as well as other players in the atherohepatic circulation, have become drug target in treating cholestasis, MESH, as well as HCC. For example, we know FXR agonist, OCA, has been studied for the last 10 years to be potentially a MESH target. But this FDA has basically denied OCA to treat MESH, despite its being approved to treat cholestasis. And Aspec inhibitors has been shown to improve glucose homeostasis, as well as cholestasis. And then FGF receptor inhibitors are now used to treat HCC. However, with all these FXR modulators, there is clear beneficial effects, such as increased insulin sensitivity, reduced inflammation. However, they all come with some side effects, including increased LDL associated with pruritus, or in some cases, severe liver injury. So to further study what could be the side effects of increased FXR activation, we made the FGF15 transgenic mice. After RNA-seq analysis of biotype FGF15 intestine-specific knockout and FGF15 transgenic mice, we found out in addition to reducing bioassay synthesis, there's other genes, especially in phase 1 P415 pathways. Some genes are highly induced. For example, CYP2B9, CYP2B10, CYP17A1 are all highly induced in FGF15 transgenic mice. So when we talk about CYP2B9, CYP3A11, first thing is we think they are associated with the antibiotic nuclear receptors like CAR and the PXR. By looking more closely to those pathways, we think it's more CAR, not PXR, because there are more CAR-specific targets are being induced rather than PXR. So in collaboration with Dr. Wen Xie, Xiao Chaoma, and then Hao Jiezhu, and then Wen Binghuang's lab, we made the FGF15 transgenic and CAR knockout mice. So, but we found out that CAR activation is not the pathway to cause all those changes. For example, this is the FGF15 transgenic and then CAR double knockout mice. Some of the induced genes, like 2B10 and then AKR1B7, does not really reduce after the knocking out CAR. So in addition, when we try to use the in vitro model, so treated a CAR overexpressed cell line with FGF19, the human version of FGF15, we did not really see CAR activation in the cells. In contrast, some of the CAR activators, like Phenobar or Sitco, can clearly cause CAR nuclear translocation. So this is a collaborative study with Wen Binghuang's lab. So therefore, we were puzzled, so what's the underlying mechanism? When we look more closely to the gene set, we notice that all the female-dominated genes are overly induced, whereas the male-dominated genes are suppressed. So we then start looking at, what are the mechanisms causing this gender switch? So the mice we used were male mice. And then, so we start looking at the literature, and one pathway that is very specific is male mice and female mice. In addition, they are regulated by sex hormones, like testosterone, estrogen. They're actually regulated by growth hormone secretion pattern. We know in males, there is a search for growth hormone, but in females, there is not a search for growth hormone. Then we start looking at our mice, and we've noticed that the mice, indeed, not only leaner, but they're also smaller. And also, they're proportionally smaller in that they don't really have a significant job in some organ weight, such as fat or muscle. The density actually is proportional to wild-type mice, but the mice are just leaner and smaller, and this is very much similar to the growth hormone-deficient mice. Then we start measuring growth hormone levels in the mice, as well as a growth hormone target gene, insulin-like growth factor 1. So on the left, you will see the red lines indicate wild-type mice, and then the blue lines are the transgenic mice. The top is... the left top is male mice, and then the right is female mice. So female mice, we know, don't really have growth hormone search. So therefore, there's no major significance between the wild-type and the transgenic mice. But for male mice, we can clearly tell that the growth hormone search is diminished in the transgenic mice. When we measured the insulin growth factor 1, both in male as well as in female mice, once FGF15 is overexpressed, the FGF... the growth hormone target gene insulin-like growth factor protein levels are dropped, indicating growth hormone pathways are diminished. So what are the growth hormone signaling in the liver? Is growth hormone actually activated JAK2 and the STAT5 pathway to induce all those growth hormone target genes? Indeed, in our transgenic mice, we noticed that the phosphorylation of PSAT5 and then to a certain degree PSAT3 are reduced, indicating that FGF15 overexpression can reduce growth hormone signaling. So then, the pathway we propose is, when the mice have increased FGF15, there is a reduced bioassay synthesis, and therefore, lipid absorption will be reduced. And then this leads to a nutrient deficiency state in the mice. And that will cause growth hormone deficiency and then reduce growth hormone signaling. This phenomenon has also been vitally reported in humans, especially in Africa. There's a lot of children suffering from nutrient deficiency, basically either it's total nutrient deficiency or just high carb, low protein, low lipid. And then the growth hormone pathways are diminished in humans, too. In addition, this is just reported last year. We know OCA, the synthetic chenodeoxycholic acid, is reported to be the first MASH target to treat human MASH disease. However, after OCA was used in some cholestatic patients, like PPC patients, there is acute liver injury regarding the use of OCA. This is basically a chart reported by FDA. So in patients after using OCA, there is a huge surge of DLE case, indicating that in some patients, OCA actually caused severe liver injury rather than leading to beneficial outcomes. So OK, coming back is, so in addition, OCA can reduce bioassay synthesis. Here is a major FXR target in the liver, is BSAP. So we think it's in some of the cholestatic patients, especially severe cholestasis like PVC, activating FXR will lead to a big induction of BSAP, especially in human patients. In this case, if you try to pump more bioassays into a blocked bioduct, therefore, that's going to end up with very acute cholestasis in PVC patients rather than have a long-term beneficial effect. I think this phenomenon has been also manifested in another model like MDR-2 knockout mice. So not only OCA has very severe liver toxicity in severe cholestatic patients, in our work, we also reported that erythrodeoxycholic acid can also work FXR agonist in the intestine. We know UDCA is another agent that's used to treat cholestasis, and in contrast to OCA has a widespread report of liver injury. CDCA has also reported to have liver toxicities, especially in PVC patients, but this has not been really put into a big emphasis in the clinic. So in our hands, using the double knockout mice, which has very low bioacid production in endogenously, we showed that erythrodeoxycholic acid can also work as FXR agonist, especially in the intestine, rather than just an FXR antagonist. So this work was maybe carried out by my talented students, Rulai He and the scientists' board. So all this evidence clearly suggests that whole-body FXR is less suitable as a target for mesh treatment. Therefore, it was not a surprise that OCA was not approved by FDA as a mesh model. In addition, other pharmaceutical companies also identified species-specific side effects of FXR ligands. In addition to the ones we just mentioned, like increased LDL, pruritus, there's also not reported is a bone abnormality in rats. Rats have very, very severe osteoporosis after FXR ligand treatment. So this can all lead to potential adverse effects if we keep on developing whole-body FXR ligand. As we mentioned early on, just like for the last few months or two years, sulfated bioacids or some FXR agonists can cause itchy symptoms. And these are maybe due to activation of this G-protein-coupled receptor HX4. This is also hard to study in mice because, number one, mice do not really have this itchy pathway. Number two, sulfated bioacids are mainly a human phenomenon, and the mice do not have so much sulfated bioacids. So this made us prompt to study what are the cell-specific FXR effects. So this is another PhD project by my talented graduate student, Sakaya Hendry, who's now a postdoc at NIHS. She started using either liver- or intestine-specific FXR knockout mice. And then, actually, this project was studied a long time ago by a postdoc, Dr. Laurie Armstrong. She's now working in a pharmaceutical company. And then when we fed the tissue-specific FXR knockout mice with either high-fat or recently the NASH diet. So Laura's data indicate that if you don't have FXR in... This is albumin FXR knockout mice. So FXR is deleted either in hepatocytes or cholangiocytes. There is increased toxicity in both lipid, liver injury, ALT, and then this also associated with a time-dependent increase of inflammation and fibrotic markers. So we know recently there is a switch used from high-fat diet to the NASH diet because in this model we can see more clearly fibrosis. So Sakaya started feeding the tissue-specific knockout mice with NASH diet. And then also she reported that if the mice are on NASH diet, there is an increased liver injury in the liver-specific FXR knockout mice in contrast to the wild-type mice as well as to the intestine-specific knockout mice. Interestingly, this can only be seen in females, and we don't really see that in males anymore. We think this is probably another sex-specific differences we should look in the future is male and female might respond to lipid versus sugar differentially. Maybe male is more sensitive to lipid toxicity versus females are more susceptible to cholesterol and sugar toxicity. Again, but nonetheless, her data and Laurel's data indicate that the liver FXR seems to be more important in regulating inflammation as well as fibrosis in contrast to the intestine FXR, which actually in many ways has a protective mechanism other than detrimental effect. In addition to hepatocytes, we also look at the role of FXR in hepatic starting cells. So we reported that FXR may actually provide a protective role in hepatic starting cells. This was founded by cell number one. When we use an increased bioassay model, FGF15 knockout mice, we actually see less fibrosis despite these mice still develop steatosis. In addition, we found out that the mechanism seems like FGF15 does not really directly suppress HSC activation, but they suppress their proliferation. And then the increased bioassays may activate FXR in hepatic starting cells to provide the deactivation. And this was being further confirmed by a paper published last year showing that hepatic starting cells actually highly express FXR, and then FXR expression is required for HSCs to maintain quiescent stage. So now is really the time to study tissue-specific FXR. So this is a study we reported actually more than a decade ago, showing that despite FXRs highly expressed in the liver and intestine, there is a huge difference between the intestine FXR versus the liver FXR in regulating differential pathways. So two years ago, one of our postdocs wrote a review article, highlighted the known factors or co-factors associated with FXR in the liver versus intestine, and we can say there is quite a difference between the reported co-activators or co-repressors associated with FXR in the liver versus that in the intestine. There's basically very little information about intestine. So in following all these reports, this is unpublished data we just completed last month. We did a FXR RIME assay, so basically pulled out FXR associated with the genome and sent to a proteomic analysis in both the FXRs in HFG2 cells representing liver and HT29 cells representing intestine. So we tried to use a purified system to start with. To our surprise, we actually pulled out more than 2,000 factors in each cell line, and then many of the clusters indicating not only those factors are associated with chromatin remodeling, co-transcriptional activation, or inhibition, there's other factors associated with FXR, like ribosomal proteins, a lot of ligase, actually protein degradation, as well as nucleosomal homeostasis. So indicating that our concept about FXR's functions are still way too simplified. There is a lot more functions of FXR, or perhaps this could apply to other nuclear receptors that we do not know yet. So in summary, homeostasis of bile acids is tightly linked to physiology and pathology, and animal models are evolving but still not there yet. One thing I want to point out is some industries start using other animal models like hamster to study FXR and bile acid pathways. Hamster is also not perfect, but they are falling somewhere in between human or non-human private and rodent. So in that regard, hamster may be a useful model in the future as a supplemental model to study disease and the bile acid pathway. FXR is critical for liver functions, but targeting systemic FXR may not be a good strategy in disease treatment. And then there's an urgent need in understanding cell-specific activation or maybe inhibition of FXR mechanisms. And then there's also an urgent need to understand individual bile acid functions in regulating physiology and the contribution to pathology. And then again, all this work cannot be done with my talented trainees in the past, as well as many, many collaborators, including Jim, Lauren, Xiaobo, Wenxie, Dr. Buckley here, Dr. Shuo, Dr. Shuo Xiao, Yihong Li, my older mentors, Dr. Carson, Dr. Gonzalez, Dr. Chen, and then Dr. Jiang. With that, I'll stop here and welcome any questions you may have. Thanks, Grace. Yeah, that was great. That's a lot. As a reminder to those, if you have questions, if you can put those into the chat, you can either type out your question or you can say, I have a question, and we will do our best to call upon you in the order that questions are received, and you can just unmute and ask your question. So I think while those are coming in, I get the traditional conference thing. I have a comment, and that leads to a question, and that is, the comment is, every time I try to think about all of the moving parts associated with this, I get increasingly lost and realize what I really don't know is a lot. And I'm getting the impression that every time an experiment gets done in the field, one experiment gets us 10 new questions on the connections that are—and if you want to elaborate on that point, that would be great, but my question is this, and this is not us at the moment, but for investigators that are looking to jump into this, with all of the moving parts associated with the microbiome and bile acids and all of that, how does one begin to control the moving parts associated with microbiome-mediated metabolism of bile acids within a standard mouse colony? I mean, the husbandry is challenging enough, and then we look to then replicate experiments across institutions. How do you advise someone who wants to start on that? That's a great question, Jim. I have exactly the same feeling. The more I know about bile acids and LXR, I feel like I have so much unknowns. But I think there's a way to do this. You could dissect that. Number one good thing is bile acid homeostasis are quite stable. The bile acid pools are stable in many ways, and then the species compositions are quite stable, and the atherohepatic circulation is the major regulating factor for bile acid homeostasis. And then in mice, because the CYP2A12 expression, the impact on liver by the secondary bile acids are less severe unless you have really severe disturbance or injury. So you think about secondary bile acids. Otherwise, like LCA will be secreted through fecal excretion. DCA will be converted to CA in the liver in mice. But in humans, there is a huge interest in finding correlations between DCA versus disease because we humans don't have 2A12. So remember Alan Hoffman, who is a bile acid researcher in South San Diego, passed away a few years ago, basically is a bile acid legend. He profiled human DCA and found out that DCA increasingly accumulates in our body with aging, like when people are aged like 60s, 70s. Up to 65% to 70% of the circulating bile acids are DCA rather than other bile acids. So in that regard, like the 2A12 knockout mice could be actually a very interesting tool in the future, especially liver-related diseases. It just needs to systematically look at those factors and then look at correlations with other pathways. There's always some insights to learn, although not perfect, definitely not perfect, but there's always some insight to learn. Like personally, myself, try to be shy away from microbiome because it's too complicated. So I'm using secondary bile acids as a readout if there's any functions in mice. Yeah, it's never a perfect model, but I think I'm like positive, beginning a bit more knowledge each day, no matter what model you're using. I like it. Thanks much. Lily? I have a clinical question and then kind of like a basic science angle to it. I have a question about OCA. So I see a lot of PBC in my clinic. And as you know, I'm interested in hepatotoxicity. So this is where my interests intersect, although I don't study bile acids specifically. Just last week, for example, I had a patient who was started on UDCA and had severe itch. And I have patients whose itch gets better on UDCA. Obviously, UDCA is a color radic. And I have patients that randomly have really worsening of their itch with UDCA. So the fact that you bring up the 3-sulfated form, and I know UDCA can also go through that pathway, is that why you think possibly some patients, does everyone basically form the 3-sulfate UDCA or is that, you think, unique to some patients? And why do some patients itch and some don't? That's a brilliant question. Just from my perspective, I think there's probably two, at least two underlying things with this question. Number one is PBC patients, I don't know in clinic how you assess like binary abstraction. Probably some patients are more abstracted than the other. Abstracted than the other. Like my thinking is just like a blocking a pipe, like a irritation pipe. If the atrial hepatic circulation is circulating, either OCAL or UDCA probably are good candidates in reducing cholestasis because they really suppress bile acids and improving the atrial hepatic circulation. And they're forced, like analogous to make a water start running in the water system. But I don't know in the clinic, do you have a message? So yeah, so this is like a non-serotic, early PBC patient, ALK-Fox at 700, right? In humans, normal is about 100. Normal bilirubin, normal excretory function, bilirubin is like 0.4, gets started on UDCA and starts itching. So I like this and I thank you for introducing me to that cell paper. I downloaded it as you were speaking, I'm going to read about it. So I was just thinking that maybe there's something, if we could measure UDCA 3-sulfate metabolites, maybe some people form more 3-sulfate metabolites versus others. And that's why some people itch and some people don't. So I don't know if there's any data on that, but it would be very interesting if we have like prospective serum collected, which I know a lot of the PBC banks do to see if that's a consideration. The other thing I was going to say is that with the, and ask you about, and it's related, with obesicolic acid, one of my theories, I've never been able to prove this, is that, you know, obviously there was more toxicity associated with the NASH when it was used for MASH. And one of my theories for that was that obviously the dose was different for the two models, but even when you looked at the 10 milligram dose in the MASH model. And I think that UDCA, because in the PBC patients, they also got UDCA, the UDCA had a protective effect in that because it's a coloretic, when you combine, you know, that this potent FXR agonist with UDCA, you're causing coloresis and you're kind of improving flow. And that's, I feel like that's kind of what you were saying. So there's no head-to-head comparison of like toxicity of OCA alone versus OCA plus UDCA. But I think that UDCA was the saving grace in PBC in that it was helping the low-dose OCA get colorest better. Does that make sense? I agree. I think like probably keeping atherohepatic flow is more important in regulating like net bio-acid concentration and levels. Like we know in females versus male, women has way more bio-acids. This is also true in mice. But as long as bio-acids are perfectly transporting throughout the atherohepatic system, it's fine. It's only when problem happens when they are stopped, the transporting, like cholestasis or leaked into other parts of the body. And I agree with you. When the bilirubin is elevated, that's a sign that they're not going to do well because they're already showing that their excretory function is diminished, right? So I think the bilirubin... Exactly. Yeah, exactly. If the patients do not really have bilinear blockage, then they probably will be benefited from... But if they have... Yeah, if the transport is blocked, then I think that's really... Because like I mentioned, BSAP is the major bio-acid transporter in humans. And then they are highly induced by FXR activation. And OCA is pretty potent, yeah. Very potent in inducing BSAP, yeah. And then for sulfated bio-acids, there are two stories. One, the microbiome contributes to the production. The other is, especially in female, female has a higher expression of sulfation, the sulfotransferases. So therefore, this is probably why in female patients, sulfated bio-acids actually has a high... In general, should have a higher level compared to male patients. That's very interesting. You've given me some ideas for a little translational study. We have more questions for you. All right. So Dr. Cordoba, do you want to unmute and ask your question? I'll give you a moment. Yeah, yeah, sure. It's a great presentation, Dr. Guo. So my question was related about the story of fatigue and growth hormone. And it was if you guys have seen any direct effect of FGF-15 on the pituitary or the hypothalamus? And maybe it's like that brain gut axis? Yeah, that's a good question. No, we don't. We never looked anything beyond the lower abdominal cavity yet. But FGF-15, we know, especially in my team, works on some of the neurons. I think I've forgotten the name. Basically, they regulate energy homeostasis quite potently. But on pituitaries as well as hypothalamus, I have no idea whether they have effects or not. Yeah, because it's seen that the decrease in growth hormone levels, I mean, it's a major thing. So I would expect that this is hitting the neurons in the hypothalamus. And also, if you have, if they have reduced growth hormone input in the liver, will they be more susceptible to NASH or FGF-15 may be protective in that way and it's counteracting? That's a good question. Actually, we did the study. The paper is not published. We don't know the mechanism. We don't know the mechanism. But for the transgenic mice, once you put them onto either high-fat or the NASH diet, they're completely protected from, like, steatosis. The lower was very, very lean. So we think the thing is could be due to, number one, they don't have bioassays. So the dietary lipid absorption will be decreased. And number two, we know FGF-15 and the 19 really promoting energy expenditure by central system as well as in the brain. But on the other hand, in those mice, even they have very lean liver, but we do see increased inflammation, especially like portal inflammation. So indicating FGF-15, 19 to reduce liver fat may still has to be cautious. Thank you. Sure. Awesome. So next up, Dr. Kambu, would you like to unmute and ask your question? If not, I got you covered. Sure. Thank you. Very nice presentation, Dr. Gu as well. So my question is, like, it does seems like there is a tissue or cell-specific effect of FXR. So in that aspect, like, do you think the FXR isoforms could account for that effect? That's a very good question. So we know there's FXR alpha-1, alpha-2, beta-1, beta-2, and it seems differentially expressed in the liver versus intestine. Nobody has ever looked at that aspect. All four isoforms, this was actually a brilliant study by Yanqiao almost 20 years ago, seems to be activated by CDCA quite well. But whether they're responsible for getting access to the target genes in the chromatin in different cell types, we don't know. And then, so one interesting thing is, like, say adrenal, FXR is highly expressed in the adrenal. And our RNA-seq data indicating, like, FXR do affect adrenal gene expression, maybe related to the steroid hormone-related production. But who's activating FXR in the adrenal? Don't have bile acids there. So there's a lot of questions left without good answers right now, just looking at bile acids. Thank you. Sure. Thanks. Any other last-minute questions? Coming up on noon. Going once. Looking. Going twice. Oh, okay. So then I have a few announcements. The first one is to thank Dr. Guo for a great talk. That was wonderful. And then coming with that announcement is that these talks are recorded, right? So you all can come back and access this later, because when you get a presentation that has that much data and depth of mechanism and kind of future study thinking, you may want to come back to access that. And we would encourage you to do that. That is arranged by another person we owe our thanks to, and that's Julie Hoffman, who takes care of setting this up for us, as well as the recordings. I think that's about it. So we'll wrap up for today. Grace, thanks again. That was really cool. And thanks to all for attending. Thank you for your flexibility for doing this in February. We much appreciate it, and we really enjoyed the talk. Yeah. Thank you all. Thanks all. Bye.

bile acids

cell signaling

cytotoxicity

FXR receptors

liver diseases

animal models

species differences

therapeutic strategies

genetically modified mice