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In the last decade, research into human hepatology has been revolutionized by the development of mini human livers in a dish. These liver organoids are formed by self-organizing stem cells and resemble their native counterparts in cellular content, multicellular architecture, and functional features. Liver organoids can be derived from the liver tissue or pluripotent stem cells generated from a skin biopsy, blood cells, or renal epithelial cells present in urine. With the development of liver organoids, a large part of previous hurdles in modeling the human liver is likely to be solved, enabling possibilities to better model liver disease, improve (personalized) drug testing, and advance bioengineering options. In this review, we address strategies to generate and use organoids in human liver disease modeling, followed by a discussion of their potential application in drug development and therapeutics, as well as their strengths and limitations., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background & Aims: The nuclear receptor subfamily 1 group H member 4 (NR1H4, also called FXR) is a ligand-activated transcription factor that, upon binding of bile acids, regulates the expression of genes involved in bile acid, fat, sugar, and amino acid metabolism. Transcript variants encode the FXR isoforms alpha 1, alpha 2, alpha 3, and alpha 4, which activate different genes that regulate metabolism. Little is known about the mechanisms by which the different isoforms regulate specific genes or how the expression of these genes affects the outcomes of patients given drugs that target FXR., Methods: We determined genome-wide binding of FXR isoforms in mouse liver organoids that express individual FXR isoforms using chromatin immunoprecipitation, followed by sequencing analysis and DNA motif discovery. We validated regulatory DNA sequences by mobility shift assays and with luciferase reporters using mouse and human FXR isoforms. We analyzed mouse liver organoids and HepG2 cells that expressed the FXR isoforms using chromatin immunoprecipitation, quantitative polymerase chain reaction, and immunoblot assays. Organoids were analyzed for mitochondrial respiration, lipid droplet content, and triglyceride excretion. We used the FXR ligand obeticholic acid to induce FXR activity in organoids, cell lines, and mice. We collected data on the binding of FXR in mouse liver and the expression levels of FXR isoforms and gene targets in human liver tissue and primary human hepatocytes from the Gene Expression Omnibus., Results: In mouse liver cells, 89% of sites that bound FXR were bound by only FXRα2 or FXRα4, via direct interactions with the DNA sequence motif ER-2. Via DNA binding, these isoforms regulated metabolic functions in liver cells, including carbon metabolism and lipogenesis. Incubation with obeticholic acid increased mitochondrial pyruvate transport and reduced insulin-induced lipogenesis in organoids that expressed FXRα2 but not FXRα1. In human liver tissues, levels of FXRα2 varied significantly and correlated with expression of genes predicted to be regulated via an ER-2 motif., Conclusions: Most metabolic effects regulated by FXR in mouse and human liver cells are regulated by the FXRα2 isoform via specific binding to ER-2 motifs. The expression level of FXRα2 in liver might be used to predict responses of patients to treatment with FXR agonists., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The nuclear receptor Farnesoid X Receptor (FXR) is activated by bile acids and controls multiple metabolic processes, including bile acid, lipid, carbohydrate, amino acid and energy metabolism. Vitamin A is needed for proper metabolic and immune control and requires bile acids for efficient intestinal absorption and storage in the liver. Here, we analyzed whether FXR regulates vitamin A metabolism. Compared to control animals, FXR-null mice showed strongly reduced (>90%) hepatic levels of retinol and retinyl palmitate and a significant reduction in lecithin retinol acyltransferase (LRAT), the enzyme responsible for hepatic vitamin A storage. Hepatic reintroduction of FXR in FXR-null mice induced vitamin A storage in the liver. Hepatic vitamin A levels were normal in intestine-specific FXR-null mice. Obeticholic acid (OCA, 3 weeks) treatment rapidly reduced (>60%) hepatic retinyl palmitate levels in mice, concurrent with strongly increased retinol levels (>5-fold). Similar, but milder effects were observed in cholic acid (12 weeks)-treated mice. OCA did not change hepatic LRAT protein levels, but strongly reduced all enzymes involved in hepatic retinyl ester hydrolysis, involving mostly post-transcriptional mechanisms. In conclusion, vitamin A metabolism in the mouse liver heavily depends on the FXR and FXR-targeted therapies may be prone to cause vitamin A-related pathologies.

Objective: Coupling metabolic and reproductive pathways is essential for the survival of species. However, the functions of steroidogenic enzymes expressed in metabolic tissues are largely unknown., Methods and Results: Here, we show that in the liver, the classical steroidogenic enzyme Cyp17a1 forms an essential nexus for glucose and ketone metabolism during feed-fast cycles. Both gain- and loss-of-function approaches are used to show that hepatic Cyp17a1 is induced by fasting, catalyzes the production of at least one hormone-ligand (DHEA) for the nuclear receptor PPARα, and is ultimately required for maintaining euglycemia and ketogenesis during nutrient deprivation. The feedback-loop that terminates Cyp17a1-PPARα activity, and re-establishes anabolic liver metabolism during re-feeding is mapped to postprandial bile acid-signaling, involving the receptors FXR, SHP and LRH-1., Conclusions: Together, these findings represent a novel paradigm of homeostatic control in which nutritional cues feed-forward on to metabolic pathways by influencing extragonadal steroidogenesis., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The Farnesoid X receptor (FXR) is a nuclear receptor activated by bile acids (BAs). BAs are amphipathic molecules that serve as fat solubilizers in the intestine under postprandial conditions. In the post-absorptive state, BAs bind FXR in the hepatocytes, which in turn provides feedback signals on BA synthesis and transport and regulates lipid, glucose and amino acid metabolism. Therefore, FXR acts as a homeostat of all three classes of nutrients, fats, sugars and proteins. Here we re-analyze the function of FXR in the perspective of nutritional metabolism, and discuss the role of FXR in liver energy homeostasis in postprandial, post-absorptive and fasting/starvation states. FXR, by regulating nutritional metabolism, represses autophagy in conditions of nutrient abundance, and controls the metabolic needs of proliferative cells. In addition, FXR regulates inflammation via direct effects and via its impact on nutrient metabolism. These functions indicate that FXR is an attractive therapeutic target for liver diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background & Aims: The nuclear receptor subfamily 1 group H member 4 (NR1H4 or farnesoid X receptor [FXR]) regulates bile acid synthesis, transport, and catabolism. FXR also regulates postprandial lipid and glucose metabolism. We performed quantitative proteomic analyses of liver tissues from mice to evaluate these functions and investigate whether FXR regulates amino acid metabolism., Methods: To study the role of FXR in mouse liver, we used mice with a disruption of Nr1h4 (FXR-knockout mice) and compared them with floxed control mice. Mice were gavaged with the FXR agonist obeticholic acid or vehicle for 11 days. Proteome analyses, as well as targeted metabolomics and chromatin immunoprecipitation, were performed on the livers of these mice. Primary rat hepatocytes were used to validate the role of FXR in amino acid catabolism by gene expression and metabolomics studies. Finally, control mice and mice with liver-specific disruption of Nr1h4 (liver FXR-knockout mice) were re-fed with a high-protein diet after 6 hours fasting and gavaged a 15 NH 4 Cl tracer. Gene expression and the metabolome were studied in the livers and plasma from these mice., Results: In livers of control mice and primary rat hepatocytes, activation of FXR with obeticholic acid increased expression of proteins that regulate amino acid degradation, ureagenesis, and glutamine synthesis. We found FXR to bind to regulatory sites of genes encoding these proteins in control livers. Liver tissues from FXR-knockout mice had reduced expression of urea cycle proteins, and accumulated precursors of ureagenesis, compared with control mice. In liver FXR-knockout mice on a high-protein diet, the plasma concentration of newly formed urea was significantly decreased compared with controls. In addition, liver FXR-knockout mice had reduced hepatic expression of enzymes that regulate ammonium detoxification compared with controls. In contrast, obeticholic acid increased expression of genes encoding enzymes involved in ureagenesis compared with vehicle in C57Bl/6 mice., Conclusions: In livers of mice, FXR regulates amino acid catabolism and detoxification of ammonium via ureagenesis and glutamine synthesis. Failure of the urea cycle and hyperammonemia are common in patients with acute and chronic liver diseases; compounds that activate FXR might promote ammonium clearance in these patients., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: The bile acid-activated farnesoid X receptor (FXR) is a nuclear receptor regulating bile acid, glucose and cholesterol homeostasis. Obeticholic acid (OCA), a promising drug for the treatment of non-alcoholic steatohepatitis (NASH) and type 2 diabetes, activates FXR. Mouse studies demonstrated that FXR activation by OCA alters hepatic expression of many genes. However, no data are available on the effects of OCA in the human liver. Here we generated gene expression profiles in human precision cut liver slices (hPCLS) after treatment with OCA., Methods: hPCLS were incubated with OCA for 24 h. Wild-type or FXR(-/-) mice received OCA or vehicle by oral gavage for 7 days., Results: Transcriptomic analysis showed that well-known FXR target genes, including NR0B2 (SHP), ABCB11 (BSEP), SLC51A (OSTα) and SLC51B (OSTβ), and ABCB4 (MDR3) are regulated by OCA in hPCLS. Ingenuity pathway analysis confirmed that 'FXR/RXR activation' is the most significantly changed pathway upon OCA treatment. Comparison of gene expression profiles in hPCLS and mouse livers identified 18 common potential FXR targets. ChIP-sequencing in mouse liver confirmed FXR binding to IR1 sequences of Akap13, Cgnl1, Dyrk3, Pdia5, Ppp1r3b and Tbx6., Conclusions: Our study shows that hPCLS respond to OCA treatment by upregulating well-known FXR target genes, demonstrating its suitability to study FXR-mediated gene regulation. We identified six novel bona-fide FXR target genes in both mouse and human liver. Finally, we discuss a possible explanation for changes in high or low density lipoprotein observed in NASH and primary biliary cholangitis patients treated with OCA based on the genomic expression profile in hPCLS., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)