Good morning. I'm Kathleen Corey from Harvard Medical School and Mass General Hospital, and we're thrilled to have you here. And we'd like to introduce our first speaker. So our first speaker will be John Chiang from Northeastern Ohio Medical University, who will be talking to us about the gut microbiome, nuclear hormone receptors, and liver fibrosis. Thank you. I'd like to thank the organizer for inviting me to give a talk in this SIG. I was given this title, gut microbiome, nuclear hormone receptor, and liver fibrosis, but I have to admit I'm not an expert in gut microbiome. I'm more working on the bio-acid, which has intimate relationship between the bio-acid metabolism and gut bacteria. Okay, so I have nothing to...oh, did I touch something wrong? Okay, that's fine. All right. So the outline of my talk is the bio-acid synthesis in the liver and the bio-transformation in the gut. And then I will give a very brief introduction about the role of the bio-acid activator receptor, phenosol X receptor, and TGR5, which is G-protein copper receptor, in the regulation of bio-acid homeostasis. And then we'll get into non-alcoholic fatty liver disease. And then I will give a talk of a recent study on the intestine FXR signaling in the gut microbiome in obesity and diabetes. Bio-acids are derived from cholesterol. So bio-acids are the end product of cholesterol catabolism in the liver. So bio-acid metabolism actually controls the cholesterol homeostasis because cholesterol is a substrate. And the end product are bio-acids. So in the past, the bio-acid has been considered just as a physiological detergent required for intestine absorption of nutrients and drugs and xenobiotics. Nobody can survive without bio-acids. And 20 years ago, it has been identified that bio-acid can activate the nuclear receptor FXR and also G-protein copper receptor TGR5 to regulate lipid glucose, antigen homeostasis, and to protect against obesity and diabetes. Those are fairly well established. And then bio-acid control gut bacteria overgrowth and to protect intestine barrier function. So it's a fairly important function for bio-acid in the gut. And then potential therapeutic drug for treatment of nephro has been developed recently. So the bio-acid synthesis pathway has two pathways. One is called carboxyl pathway. And another one is alternative pathway. So the main pathway is a classic pathway. It is initiated by an enzyme called C7A1, cholesterol 7-alpha hydroxylate, located in ER membrane. And then they go through this pathway. Actually, more than 17 different enzymes are involved in the pathway. So several steps later, and then they form the 7-alpha hydroxylate 4-cholesterol-3-1, or C4. So this has been serum level of C4 has been used as an indication of the bio-acid synthesis in mice or in humans. And then C4 is a common precursor to form caudic acid and the chenodialcyl caudic acids. To form caudic acid, the sterol 12-alpha hydroxylate, or CYP8B1, is required. Without that, the product is a chenodialcyl caudic acid there. There are many, many enzymes involved in this pathway. Sterol 27-hydroxylate is mitochondrial enzyme. It catalyzes the side chain oxidation, and then followed by paroxysomal beta oxidation enzyme to remove 3-carbon molecule to form C24 bio-acids. And then after that, bio-acids are conjugated immediately to the taurine or glycine conjugated bio-acids. And the SU-2 enzyme called bio-sul-CoA synthetase and the bio-acid aminotransferase and so on. The alternative pathway, it started from the side chain modification first. So mitochondria has very little cholesterol. D1 can transfer cholesterol into the mitochondria, and the CYP27A1 starts the first reaction to form 27-hydroxyl cholesterol. There's another 7-alpha hydroxylate called CYP7B1 involved in this process. It also produces oxysteroids. The difference between man and human is in mouse, liver, or rodent, the rats, has a C2C70. 2C70 can convert chenodioxycholic acid to alpha-muricolic acid or beta-muricolic acid. Those are highly hydrophilic bio-acids, but human does not have those bio-acids. Human has very hydrophilic bio-acids, chenodioxycholic acid. So bio-acids store in gallbladders, and after each meal, bio-acids are released to the intestine tract, and the gut bacteria modify those bio-acids. So the bio-acids are in the liver called primary bio-acids, and then when they get into the intestines, the bacterial bio-acid hydrolase deconjugates the bio-acid to free bio-acid cholic acid and chenodioxycholic acid, and then bacterial 7-alpha dehydroxylates remove a hydroxyl group from this bio-acid to form dioxycholic acid and lithocholic acid. Those are highly hydrophobic toxic bio-acids. And the bacteria also has 7-alpha-beta-hydroxy-stero-dehydroxyl activity, which can convert chenodioxycholic acid to ortho-dioxycholic acid. So you can see, changing from 7-alpha to 7-beta hydroxyl position make a highly hydrophobic bio-acid to a hydrophilic bio-acid there. So you can see in human bio-acid pool, most bio-acids are cholic acid, chenodioxycholic acid, dioxycholic acid, lithocholic acid. Those are hydrophobic bio-acids. But in mice, most bio-acids are muricolic acid, ortho-dioxycholic acid, and so on. So there's a difference between the bio-acid hydrophobicity between mice and humans. So this is a complicated slide to show you how the bio-acid feedback regulates through enterohepatic circulation to regulate the bio-acid synthesis and maintain homeostasis. So in the liver, the FXR is activated by chenodioxycholic acid or cholic acid to synthesize a repressor called SHIP. SHIP then indirectly inhibits CYP7A1. So this is a classic feedback regulation. The end product of the pathway inhibits the first step of the reaction there. FXR also plays a role in inducing BSAP, which is a bio-acid export transporter, to transport bio-acid immediately into the bio. And then mixed with cholesterol, phospholipid, to form mixed micelles. And then the bio-acid then released to the intestine tract, goes through the bio-duct, and then in the intestine, there's ASBT, the bio-salt uptake transporter, to transport bio-acid into the enterocytes and then activate the FXR. And then FXR also can induce the so-called OST alpha beta, which will efflux bio-acid into the portal circulation back to liver to complete the circulation between liver and the intestine. More recent study indication that FXR induce a FGF19 in human or FGF15 in mice. And those are important fiber growth factor. This play important role in regulation of metabolism and bio-acid metabolism. So FGF19 goes through portal circulation, goes to liver, activate a receptor 4, a beta-closal complex. And then this activate a MAP kinase, most likely through ERG pathway, and to inhibit CYP7A1. So there's a liver to intestine access to regulate this metabolism. In the air cell, FXR and TGR5 both are expressed there. So we have data to show activation FXR can induce TGR5. And the TGR5 play a role in secretion of GLP1. And GLP1 get into beta cell and stimulate insulin secretion, improve insulin sensitivity. Non-alcoholic fatty liver disease, I think you all know this quite well. I just very briefly described there's a spectrum of the phenotype from simple steatosis to NASH to cirrhosis and then make small number of percent of patient develop hepatocellular carcinoma. Simple steatosis is no problem, can be reversed. But they can be advanced to inflammatory NASH. So there's a so-called two-heat theory. The high-fat diet, obesity, and the dysbiosis can cause inflammation. And then they require second heat, like insulin resistance. The serofat, lipotoxicity, oxidative stress, and cholesterol can promote inflammation, activate the steatocell to develop fibrosis. So this is current theory about it. But there are probably other factors also involved in this process. Okay, here's the introduction about bio-acid and gut microbiota. In human feces, 90% of bio-acid belong to the fila, firmicules, or baculoidis. A higher ratio of firmicules to baculoidis can enable the gut bacteria to extract energy more efficiently from the fat from the diet, therefore can increase adiposity and also the obesity. The bio-salt hydrates its important enzyme. The activity is fairly high in firmicules, including clostridium enterococcus, and bifidobacterium, listerium, and lactobacillus, and also in baculoidis, can play a critical role in the bio-acid metabolism. It has been reported that if you overexpress bio-salt hydrates activity in mice, can reduce weight, can also reduce plasma cholesterol and liver triglyceride by inducing liver and intestine genes involved in this process, and also circadian rhythm. However, other studies say that inhibition of bio-salt hydrates activity can decrease the tauromiricotic acid. So tauromiricotic acid has been identified as the FXR antagonist in the intestine. So they can antagonize intestine FXR activity, and therefore stimulate bio-acid synthesis and can prevent the diet-induced obesity. Okay. Alright. Okay. And then inhibition of intestine FXR can improve metabolism. So there are serious studies reported by Saying et al. They find out that in germ-free mice, tauro-beta-miricotic acid level is increased, can antagonize intestine FXR, therefore can reduce FGF15, therefore can stimulate bio-acid synthesis in liver. So increasing bio-acid poor sites. And this process is FXR-dependent. And from Gonzales' laboratory, report several studies to show that antioxidant temper can reduce lactobacillus. Lactobacillus has high BSH activity, therefore also increasing tauromiricotic acid to antagonize intestine FXR and prevent DIO. And he followed by the intestine-specific FXR NAGL mice, and find out that those mice are protected from DIO and diabetes by reducing the seramide and the hepatic ER stress and always improving insulin sensitivities there. And a more recent study, the diabetic drug mefonine can reduce bacterial flagella, increasing the tauro-glycine UDCA. So UDCA in this study has been identified as an antagonist of intestinal FXR in human patients, improved metabolism. Okay, but in contrast, Ron Evans' laboratory reported that an intestinal restricted FXR agonist called fexamine is a very potent FXR agonist, can increase in FGF-15, promote higher adipotisial browning, can reduce weight, and improve glucose and insulin tolerance in DIL mice. So we tried to find out what is the mechanism. So we published a paper in Hepatology in 2018. So we garage the mice with fexamine, like 25 mg per kg body weight for 7 days, and we measured the GLP-1 secretion rate. So GLP-1 secretion rate is increased in the diabetics mice treated with fexamine. Okay, and then glucose tolerance test and insulin tolerance test all show improvement of the glucose and insulin sensitivities. And this compound is restricted in the intestine because they are not soluble, very insoluble. So they can induce the FXR, target sheep, FGF-15, OSV, alpha, beta, and so on. So it is indeed a intestine-specific agonist. And then we measure the adipotisial browning in DBDB mice and brown adipotisial and several white adipotisial to show increasing Diodinase-2, which is a target of TGR-5, can stimulate DIO-2, and the encoupling protein 1 and the PRDM, the PGC-1 alpha, and also in the white adipotisial can induce some of this browning factor, and the staining show you that they do cause some browning, and also western blot confirm the UCP-1 level is increasing by some treatment. So we analyze the bio-acids. Bio-acid pool size does not change because it does not circulate to liver to regulate the bio-acid synthesis gene at all. So bio-acid pool in the intestine, gallbladder, and liver did not change. But the gallbladder bio-acid concentration to show dramatic increasing of tolu-lisacortic acid by more than 1,000 fold. So this is fairly toxic bio-acid, but it's not very toxic in mice because they can handle that by sulfations there. So we try to find out why tolu-lisacortic acid is increasing. So we collaborated with Frank Gonzalez and Andrew Patterson, did the 16S RNA sequencing, and here I'll show you the PCA analysis. The heat map identifies several up or down regulated gene, compare the flux-armyntrator versus the vehicle. So we identified two, confirmed the two bacteria. One is called acetaldefector, is doubled, and bacteroides also increasing a lot. And then literature search identify that these two bacteria has both 7-alpha and 7-beta dehydroxy activity. So this shows you that BSH de-conjugate by the chenodioxy first. And then isomerase can convert CdCA to UdCA, and 7-alpha dehydroxy convert CdCA to LCA. And UdCA can be 7-beta dehydroxy to form LCA. So this may explain increasing level of lisacortic acid, which is the best endogenous ligand of the TGR5. So those are the acetaldefector, is gram-positive bacteria, belong to the firmicus and the clostridia. They have high BSH and 7-alpha, 7-beta dehydroxy activities. And bacteroides is gram-negative, belong to bacteroides and also have very similar properties there. And more recently we developed So that explain, you know, these fexamine have some different effect, therefore can stimulate the lisacortic acid synthesis and activate TGR5 and reduce weight. And TGR5 is known to stimulate energy metabolism in the adipose tissues and stimulate the GLP-1 secretion. So that's my conclusion. More recently our laboratory developed an FXR and TGR5 doggable knockout mice. And because these two, FXR and TGR5, are predominant bioassay activated receptor, they play important role in regulation of metabolism and so on, as I mentioned before. So we want to see if we knock out these two important genes, what happens to the mice. Those mice actually are perfect fine. The doggable knockout mice are leaner compared with wiretie mice. They are insulin sensitive, just like wiretie mice. So this is kind of surprising to us. But in other way it may not be surprising because they produce more bioassay. Bioassay has increasing insulin sensitivity. So probably that's the explanation. So TKO mice have reduced hepatolipid and increased serine cholesterol. Actually they have less steatosis there. And TKO mice has increased bioassay pool size, double the pool size. So this is very similar to our previous publication of the CYP7A1 transgenic mice. Increasing bioassay synthesis, increasing pool, beneficial can prevent obesity and diabetes. TKO mice have increased taracolic and taradioxicolic acid because they're increasing the classical pathway, increasing 7A1 and 8B1 to stimulate more bioassay synthesis, but reduce taramuric chloride acid. So they abuse the FXR antagonist, therefore reduce FGF15 and reduce SHIP. So that's consistent with lack of the FXR. So this phenotype is very similar to FXR single knockout mice. So the conclusion is the bioassay feedback regulation is impaired. Bioassay pool size only doubled, but not increasing dramatically. And then surprise to us, we find out Western high-fat diet did not cause obesity in those mice. Actually it's not a surprise because FXR knockout mice also does not gain weight. TGF5 knockout mice also does not gain weight. And all change insulin sensitivity in the double knockout mice. So we did RNA sequencing analysis of the liver transcriptome of the wild type TGF5 knockout mice, TKO mice, FXR knockout mice, and then you can see PCA analysis show the clear separations. And the Venn diagram show you we identify those overlapping gene or unique gene in FXR versus wild type and TGF5 and TKO mice. And the top regulated pathway of the disease has been identified in those FXR, TGF5 knockout mice. So all these top regulated genes are involved in steroid biosynthesis. All these are increasing. SIBP increasing. It's involved in cholesterol synthesis. So this is really very similar to the CYP7A1 double knockout mice because double knockout mice has increasing bioassay pool and FXR regulation seems kind of impaired and so on. And they have decreased activation of the gene expression by SIBP1C which is involved in the fatty acid synthesis. So those mice actually have less fat accumulated. So cholesterol synthesis is upregulated. But we noticed that they see the liver fibrosis level actually the gene involved are increasing in the double knockout mice. And connective tissue disease also increase in these mice. So I cut out quite a few slides and so we just show you one or two pieces of data there. So western dye feeding on hepatic steatosis and fibrosis. So here I just show you the serious red staining of wild type after the western dye feeding gets steatosis. FXR knockout mice has more severe steatosis as reported before. TGR5, our laboratory public paper in hepatology two or three years ago to show that TGR5 mice actually has lower hepatic steatosis can prevent fasting induced steatosis. And double knockout mice is more similar to FXR knockout mice, but not more severe. Actually perhaps less fat accumulated. Tricone staining for fiber to use indication that the double knockout mice seems to have more severe fibrosis. And then the TGR5 knockout mice doesn't have as much. And FXR knockout mice and the wild type has some fibrosis there. So our conclusion is that bioassay signaling through FXR and TGR5 play a critical role in hepatic metabolism and homeostasis. And the intestine FXR actually play a very, very important role in regulation of hepatic metabolism and homeostasis. So the gut to liver access really very, very critical for regulation of bioassay metabolism, not only bioassay metabolism, also lipid, cholesterol, and glucose metabolism to maintain metabolic homeostasis. So it is important to maintain this homeostasis to prevent the liver diseases. So both FXR agonist and antagonist can shape the gut microbiota, but differently. And then both can assert differential effect on metabolism. Both can be beneficial effect there. So it depends on the gut microbiome effect by FXR agonist or antagonist. And so both, so, all right, and it can benefit, this is what I mentioned earlier, by shaping the gut microbiota differently. And FXR and TGR5 does crosstalk to regulate metabolic homeostasis and prevent liver-related metabolic homeostasis. So the key takeaway lesson is that bioassay signaling play a critical role in maintaining metabolic homeostasis and the gut microbiota control bioassay metabolism. So gut microbiota will control bioassay composition and also control bioassay pool size because of germ-free mice, bioassay pool size increasing compared with conventional race. And then this signaling, because they alter the bioassay composition, so different bioassay have activated the FXR, TGR5 differently, and the gut liver access is very important in metabolic regulation. And a bioassay-based drug therapy, for example, Obetic Chloric Acid, which is a very potent synthetic bioassay, is in clinical trial, has been approved for treatment of PBC and also in the third phase clinical trial for NASH and perhaps will be approved very soon and are being developed for treating metabolic diseases. Okay, finally, I'd like to acknowledge my laboratory. I have a small laboratory and my Shannon Borme, she's my lab manager in the last 10 years. Jessica Ferreira is my postdoc and research assistant professor in my lab for many, many years. And my new technician, Tricia Gilliland, is now in the picture. Important to acknowledge several previous postdocs. Preeti Pesai was my student and postdoc in the last 10 years. She is now in the Cleveland Clinic Foundation. Most importantly, my student and former postdoc, Tinggang Li, he made the most important contribution to my research in the last 15 years. And my former postdoc, Ajay Dhanapuri, is now at UConn as a postdoc. And my collaborator, Frank Gonzalez, on this study, and Andrew Patterson did a gut microbiome study and also the long-time support of my research grant by two NIH grants for 23 years and 33 years. So thank you very much for your attention. Thank you very much, Dr. Chang. We'll move on.

Dr. John Chang

Northeastern Ohio Medical University

gut microbiome

nuclear hormone receptors

liver fibrosis

bioacids

metabolic regulation