Role of Bile Acids in Liver Injury and Regeneration following Acetaminophen Overdose

Bile acids play a critical role in liver injury and regeneration, but their role in acetaminophen (APAP)–induced liver injury is not known. We tested the effect of bile acid modulation on APAP hepatotoxicity using C57BL/6 mice, which were fed a normal diet, a 2% cholestyramine (CSA)–containing diet for bile acid depletion, or a 0.2% cholic acid (CA)–containing diet for 1 week before treatment with 400 mg/kg APAP. CSA-mediated bile acid depletion resulted in significantly higher liver injury and delayed regeneration after APAP treatment. In contrast, 0.2% CA supplementation in the diet resulted in a moderate delay in progression of liver injury and significantly higher liver regeneration after APAP treatment. Either CSA-mediated bile acid depletion or CA supplementation did not affect hepatic CYP2E1 levels or glutathione depletion after APAP treatment. CSA-fed mice exhibited significantly higher activation of c-Jun N-terminal protein kinases and a significant decrease in intestinal fibroblast growth factor 15 mRNA after APAP treatment. In contrast, mice fed a 0.2% CA diet had significantly lower c-Jun N-terminal protein kinase activation and 12-fold higher fibroblast growth factor 15 mRNA in the intestines. Liver regeneration after APAP treatment was significantly faster in CA diet–fed mice after APAP administration secondary to rapid cyclin D1 induction. Taken together, these data indicate that bile acids play a critical role in both initiation and recovery of APAP-induced liver injury

Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer1, 2, 3, 4, 5. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation6, 7, 8, 9, 10. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs4, 5, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy

Role of Hepatocyte Nuclear Factor 4 alpha in Hepatocyte Proliferation

Hepatocyte Nuclear Factor 4 alpha (HNF4α) is the master regulator of hepatocyte differentiation. It is involved in the up-regulation of genes involved in many classic hepatic functions including: bile acid metabolism, xenobiotic metabolism, glucose homeostasis, lipid metabolism, coagulation factor synthesis, etc. However, the role of HNF4α in regulation of hepatocyte proliferation was not known. The primary goal of this dissertation was to investigate the role of HNF4α in the regulation of hepatocyte proliferation. In these studies we utilized two novel inducible, hepatocyte specific, HNF4α knockdown mouse models. Past models of HNF4α dificiency result in deletion within the first few weeks of birth and lead to lethality within 6-8 weeks. This makes it difficult to address a role for HNF4α in hepatocyte proliferation because the liver is still growing and differentiating within this time frame. Hepatocyte-specific deletion of HNF4α in adult mice resulted in fat accumulation (steatosis), glycogen depletion, and increased hepatocyte proliferation with a significant increase in liver/body weight ratio without complimentary liver regeneration due to hepatocyte cell death. Global gene expression analysis (microarray and RNA-Seq) revealed that a significant number of the 300+ down-regulated genes are involved in hepatic differentiation, many of which are known HNF4α targets. Interestingly, a significant number of the 500+ up-regulated genes are associated with cell proliferation and cancer. Further, a combined bioinformatics analysis of ChIP-sequencing and RNA-sequencing data indicated that a substantial number of up-regulated genes are putative HNF4α targets. We have used chromatin immunoprecipitation (ChIP) to confirm three of these targets: Ect2, Osgin1, and Hjurp. Ingenuity Pathway Analysis (IPA)-mediated functional analysis revealed the most significantly activated gene network after HNF4α deletion is regulated by c-Myc. To determine the role of HNF4α in pathogenesis of hepatocellular carcinoma (HCC), we performed the classic initiation-promotion experiment using diethylnitrosamine (DEN). Deletion of HNF4α resulted in extensive promotion of DEN-induced hepatic tumors, which were highly proliferative and less differentiated. Further, the HCC observed in HNF4α-deleted mice exhibited significant up-regulation of c-Myc and its target genes. We hypothesized that HNF4α inhibits hepatocyte proliferation by repression of target genes. One possible mechanism is that HNF4α influences the epigenetic state of a given gene to promote or inhibit gene expression. We studied various epigenetic modifications associated with gene activation and repression on a 9 kb segment of the promoter of Ect2, a validated HNF4α negative target gene also known to activate hepatocyte proliferation. ChIP analysis performed on chromatin isolated from control and HNF4α KO livers indicated that a deletion of HNF4α resulted in an epigenetic switch in histone modifications at the Ect2 promoter from an inhibited to an active state; primarily affecting acetylation of histone H3K9. These data indicate that HNF4α inhibits hepatocyte proliferation, is a potential tumor suppressor in the liver, and plays a critical role in chemical carcinogenesis. We also provide data that support the hypothesis that HNF4α may be functioning through an influence on the epigenetic state of select genes

Global gene expression changes in liver following hepatocyte nuclear factor 4 alpha deletion in adult mice

Hepatocyte nuclear factor 4 alpha (HNF4α) is known as the master regulator of hepatic differentiation, which regulates over 60% of the hepatocyte specific genes. Recent studies including this (Walesky et al. Am J Physiol Gastrointest Liver Physiol. 304:G26-37, 2013) demonstrated that HNF4α also inhibits hepatocyte proliferation via repression of pro-mitogenic genes. In this study hepatocyte specific HNF4α knockout mice were generated using 2–3 month old HNF4α-floxed mice treated with Cre recombinase under Major Urinary Protein promoter delivered in AAV8 vector (MUP-iCre-AAV8). Control mice were treated with MUP-EGFP-AAV8. Livers were isolated from control and KO mice one week after AAV8 administration and used for gene array analysis. These data revealed several new negative target genes of HNF4α, majority of which are pro-mitogeneic genes inhibited by HNF4α in adult hepatocytes

Identification of NQO2 As a Protein Target in Small Molecule Modulation of Hepatocellular Function

The utility of in vitro human disease models is mainly dependent on the availability and functional maturity of tissue-specific cell types. We have previously screened for and identified small molecules that can enhance hepatocyte function in vitro. Here, we characterize the functional effects of one of the hits, FH1, on primary human hepatocytes in vitro, and also in vivo on primary hepatocytes in a zebrafish model. Furthermore, we conducted an analogue screen to establish the structure-activity relationship of FH1. We performed affinity-purification proteomics that identified NQO2 to be a potential binding target for this small molecule, revealing a possible link between inflammatory signaling and hepatocellular function in zebrafish and human hepatocyte model systems

Functional compensation precedes recovery of tissue mass following acute liver injury

International audienceThe liver plays a central role in metabolism, protein synthesis and detoxification. It possesses unique regenerative capacity upon injury. While many factors regulating cellular proliferation during liver repair have been identified, the mechanisms by which the injured liver maintains vital functions prior to tissue recovery are unknown. Here, we identify a new phase of functional compensation following acute liver injury that occurs prior to cellular proliferation. By coupling single-cell RNA-seq with in situ transcriptional analyses in two independent murine liver injury models, we discover adaptive reprogramming to ensure expression of both injury response and core liver function genes dependent on macrophage-derived WNT/βcatenin signaling. Interestingly, transcriptional compensation is most prominent in nonproliferating cells, clearly delineating two temporally distinct phases of liver recovery. Overall, our work describes a mechanism by which the liver maintains essential physiological functions prior to cellular reconstitution and characterizes macrophage-derived WNT signals required for this compensation

A fluorescence viewer for rapid molecular assay readout in space and low-resource terrestrial environments.

Fluorescence-based assays provide sensitive and adaptable methods for point of care testing, environmental monitoring, studies of protein abundance and activity, and a wide variety of additional applications. Currently, their utility in remote and low-resource environments is limited by the need for technically complicated or expensive instruments to read out fluorescence signal. Here we describe the Genes in Space Fluorescence Viewer (GiS Viewer), a portable, durable viewer for rapid molecular assay readout that can be used to visualize fluorescence in the red and green ranges. The GiS Viewer can be used to visualize any assay run in standard PCR tubes and contains a heating element. Results are visible by eye or can be imaged with a smartphone or tablet for downstream quantification. We demonstrate the capabilities of the GiS Viewer using two case studies-detection of SARS-CoV-2 RNA using RT-LAMP and quantification of drug-induced changes in gene expression via qRT-PCR on Earth and aboard the International Space Station. We show that the GiS Viewer provides a reliable method to visualize fluorescence in space without the need to return samples to Earth and can further be used to assess the results of RT-LAMP and qRT-PCR assays on Earth

Functional compensation precedes recovery of tissue mass following acute liver injury

© 2020, The Author(s). The liver plays a central role in metabolism, protein synthesis and detoxification. It possesses unique regenerative capacity upon injury. While many factors regulating cellular proliferation during liver repair have been identified, the mechanisms by which the injured liver maintains vital functions prior to tissue recovery are unknown. Here, we identify a new phase of functional compensation following acute liver injury that occurs prior to cellular proliferation. By coupling single-cell RNA-seq with in situ transcriptional analyses in two independent murine liver injury models, we discover adaptive reprogramming to ensure expression of both injury response and core liver function genes dependent on macrophage-derived WNT/β-catenin signaling. Interestingly, transcriptional compensation is most prominent in non-proliferating cells, clearly delineating two temporally distinct phases of liver recovery. Overall, our work describes a mechanism by which the liver maintains essential physiological functions prior to cellular reconstitution and characterizes macrophage-derived WNT signals required for this compensation