Long-term culture of genome-stable bipotent stem cells from adult human liver.

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.

APOE genotype dictates lipidomic signatures in primary human hepatocytes.

Apolipoprotein E (APOE) genetic variants are most notably known for their divergent impact on the risk of developing Alzheimer's disease. While APOE genotype has been consistently shown to modulate lipid metabolism in a variety of cellular contexts, the effect of APOE alleles on the lipidome in hepatocytes is unknown. In this study, we investigated the contribution of APOE alleles to lipidomic profiles of donor-derived primary human hepatocytes from 77 subjects. Lipidomic data obtained by liquid chromatography-mass spectrometry were analyzed across ε2/ε3, ε3/ε3, and ε3/ε4 genotypes to reveal how APOE modulates lipid relative levels over age and between groups. Hepatic APOE concentration, measured by ELISA, was assessed for correlation with lipid abundance in subjects grouped as per APOE genotype and sex. APOE genotype-specific differential lipidomic signatures associated with age for multiple lipid classes but did not differ between sexes. Compared to ε2/ε3, ε3/ε4 hepatocytes had higher abundance of acylcarnitines (AC) and acylphosphatidylglycerol (AcylPG) as a class, as well as higher medium and long-chain ACs, AcylPG, phosphatidylglycerol (PG), bis(monoacylglycerol)phosphate (BMP), monoacylglycerol (MG) and diacylglycerol (DG) species. The ε3/ε4 hepatocytes also exhibited a higher abundance of medium and long-chain ACs compared to the ε3/ε3 hepatocytes. Only in the ε3/ε4 hepatocytes, APOE concentration was lower and showed a negative correlation with BMP levels, specifically in females. APOE genotype dictates a differential lipidome in primary human hepatocytes. The lipids involved suggest mitochondrial dysfunction with accompanying alterations in neutral lipid storage, reflective of a general disturbance of free fatty acid metabolism in human hepatocytes with the ε4 allele.

Mucosal-associated invariant T-cell tumor infiltration predicts long-term survival in cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a malignancy arising from biliary epithelial cells of intra- and extrahepatic bile ducts with dismal prognosis and few nonsurgical treatments available. Despite recent success in the immunotherapy-based treatment of many tumor types, this has not been successfully translated to CCA. Mucosal-associated invariant T (MAIT) cells are cytotoxic innate-like T cells highly enriched in the human liver, where they are located in close proximity to the biliary epithelium. Here, we aimed to comprehensively characterize MAIT cells in intrahepatic (iCCA) and perihilar CCA (pCCA). APPROACH AND RESULTS: Liver tissue from patients with CCA was used to study immune cells, including MAIT cells, in tumor-affected and surrounding tissue by immunohistochemistry, RNA-sequencing, and multicolor flow cytometry. The iCCA and pCCA tumor microenvironment was characterized by the presence of both cytotoxic T cells and high numbers of regulatory T cells. In contrast, MAIT cells were heterogenously lost from tumors compared to the surrounding liver tissue. This loss possibly occurred in response to increased bacterial burden within tumors. The residual intratumoral MAIT cell population exhibited phenotypic and transcriptomic alterations, but a preserved receptor repertoire for interaction with tumor cells. Finally, the high presence of MAIT cells in livers of iCCA patients predicted long-term survival in two independent cohorts and was associated with a favorable antitumor immune signature. CONCLUSIONS: MAIT cell tumor infiltration associates with favorable immunological fitness and predicts survival in CCA.

The African Liver Tissue Biorepository Consortium: Capacitating Population-Appropriate Drug Metabolism, Pharmacokinetics, and Pharmacogenetics Research in Drug Discovery and Development.

Pharmaceutical companies subject all new molecular entities to a series of in vitro metabolic characterizations that guide the selection and/or design of compounds predicted to have favorable pharmacokinetic properties in humans. Current drug metabolism research is based on liver tissue predominantly obtained from people of European origin, with limited access to tissue from people of African origin. Given the interindividual and interpopulation genomic variability in genes encoding drug-metabolizing enzymes, efficacy and safety of some drugs are poorly predicted for African populations. To address this gap, we have established the first comprehensive liver tissue biorepository inclusive of people of African origin. The African Liver Tissue Biorepository Consortium currently includes three institutions in South Africa and one in Zimbabwe, with plans to expand to other African countries. The program has collected 67 liver samples as of July 2023. DNA from the donors was genotyped for 120 variants in 46 pharmacogenes and revealed variants that are uniquely found in African populations, including the low-activity, African-specific CYP2C9*5 and *8 variants relevant to the metabolism of diclofenac. Larger liver tissue samples were used to isolate primary human hepatocytes. Viability of the hepatocytes and microsomal fractions was demonstrated by the activity of selected cytochrome P450s. This resource will be used to ensure the safety and efficacy of existing and new drugs in African populations. This will be done by characterizing compounds for properties such as drug clearance, metabolite and enzyme identification, and drug-drug and drug-gene interactions. SIGNIFICANCE STATEMENT: Standard optimization of the drug metabolism of new molecular entities in the pharmaceutical industry uses subcellular fractions such as microsomes and isolated primary hepatocytes, being done mainly with tissue from donors of European origin. Pharmacogenetics research has shown that variants in genes coding for drug-metabolizing enzymes have interindividual and interpopulation differences. We established an African liver tissue biorepository that will be useful in ensuring drug discovery and development research takes into account drug responses in people of African origin.

Brain integrity is altered by hepatic APOE ε4 in humanized-liver mice.

Liver-generated plasma apolipoprotein E (apoE) does not enter the brain but nonetheless correlates with Alzheimer's disease (AD) risk and AD biomarker levels. Carriers of APOEε4, the strongest genetic AD risk factor, exhibit lower plasma apoE and altered brain integrity already at mid-life versus non-APOEε4 carriers. Whether altered plasma liver-derived apoE or specifically an APOEε4 liver phenotype promotes neurodegeneration is unknown. Here we investigated the brains of Fah-/-, Rag2-/-, Il2rg-/- mice on the Non-Obese Diabetic (NOD) background (FRGN) with humanized-livers of an AD risk-associated APOE ε4/ε4 versus an APOE ε2/ε3 genotype. Reduced endogenous mouse apoE levels in the brains of APOE ε4/ε4 liver mice were accompanied by various changes in markers of synaptic integrity, neuroinflammation and insulin signaling. Plasma apoE4 levels were associated with unfavorable changes in several of the assessed markers. These results propose a previously unexplored role of the liver in the APOEε4-associated risk of neurodegenerative disease.

Hepatic miR-144 Drives Fumarase Activity Preventing NRF2 Activation During Obesity.

BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR-144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR-144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes.

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective.

Insights From Liver-Humanized Mice on Cholesterol Lipoprotein Metabolism and LXR-Agonist Pharmacodynamics in Humans.

BACKGROUND AND AIMS: Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species-related, physiological differences. APPROACH AND RESULTS: Fah-/- , Rag2-/- , and Il2rg-/- knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, characterized by a high ratio of low-density lipoprotein to high-density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low-density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human-like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level. CONCLUSIONS: LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.

Gene Editing Correction of a Urea Cycle Defect in Organoid Stem Cell Derived Hepatocyte-like Cells.

Urea cycle disorders are enzymopathies resulting from inherited deficiencies in any genes of the cycle. In severe cases, currently available therapies are marginally effective, with liver transplantation being the only definitive treatment. Donor liver availability can limit even this therapy. Identification of novel therapeutics for genetic-based liver diseases requires models that provide measurable hepatic functions and phenotypes. Advances in stem cell and genome editing technologies could provide models for the investigation of cell-based genetic diseases, as well as the platforms for drug discovery. This report demonstrates a practical, and widely applicable, approach that includes the successful reprogramming of somatic cells from a patient with a urea cycle defect, their genetic correction and differentiation into hepatic organoids, and the subsequent demonstration of genetic and phenotypic change in the edited cells consistent with the correction of the defect. While individually rare, there is a large number of other genetic-based liver diseases. The approach described here could be applied to a broad range and a large number of patients with these hepatic diseases where it could serve as an in vitro model, as well as identify successful strategies for corrective cell-based therapy.

Imbalance of Genes Encoding Natural Killer Immunoglobulin-Like Receptors and Human Leukocyte Antigen in Patients With Biliary Cancer.

BACKGROUND & AIMS: Bile duct tumors are rare and have poor prognoses. Natural killer (NK) cells are frequent in human liver and infiltrate these tumors but do not control their progression. Responses of NK cells are regulated by NK immunoglobulin-like receptors (KIRs), which interact with HLA class I ligands. We aimed to characterize the features of the KIR gene loci and their ligands in patients with bile duct cancer (BDC). METHODS: We performed combined multidimensional characterization of genes that encode KIRs and their ligands in blood samples from patients with BDC from Sweden, followed for up to 8 years after diagnosis (n = 148), in 2 geographically matched cohorts of healthy individuals from Northern Europe (n = 204 and n = 900), and in healthy individuals from 6 geographically unrelated populations (n = 2917). We used real-time polymerase chain reaction, RNA sequencing, immunohistochemistry, and flow cytometry to evaluate NK-cell presence, as well as KIR and KIR-ligand expression in bile duct tumors and control tissues. RESULTS: Patients with bile duct tumors had multiple alterations at the KIR gene loci. KIR loci are grouped into genotypes that encode more inhibitory (group A) and more activating (group B) receptors, which can be subdivided into centromeric and telomeric fragments. Patients with BDC had a lower prevalence of KIR2DL3, which was linked to disequilibrium in centromeric A/B and B/B genotypes, compared with control individuals. The associations between KIRs and KIR ligands differed between patients with BDC and control individuals; patients had an altered balance between activating and inhibitory KIRs. KIR-positive NK cells infiltrated biliary tumors that expressed matched KIR ligands. CONCLUSIONS: In a multidimensional analysis of DNA from blood samples of patients with BDC in Europe, we found patients to have multiple alterations at the KIR and HLA gene loci compared with control individuals. These alterations might affect NK-cell tumor surveillance. NK cells from bile duct tumors expressed KIRs and were found in tumors that expressed cognate ligands. This should be considered in development of immune-based therapies for BDC.

Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations.

BACKGROUND & AIMS: Alagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms. METHODS: Mice with a missense mutation (H268Q) in Jag1 (Jag1+/Ndr mice) were outbred to a C3H/C57bl6 background to generate a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice). Liver tissues were collected at different timepoints during development, analyzed by histology, and liver organoids were cultured and analyzed. We performed transcriptome analysis of Jag1Ndr/Ndr livers and livers from patients with Alagille syndrome, cross-referenced to the Human Protein Atlas, to identify commonly dysregulated pathways and biliary markers. We used species-specific transcriptome separation and ligand-receptor interaction assays to measure Notch signaling and the ability of JAG1Ndr to bind or activate Notch receptors. We studied signaling of JAG1 and JAG1Ndr via NOTCH 1, NOTCH2, and NOTCH3 and resulting gene expression patterns in parental and NOTCH1-expressing C2C12 cell lines. RESULTS: Jag1Ndr/Ndr mice had many features of Alagille syndrome, including eye, heart, and liver defects. Bile duct differentiation, morphogenesis, and function were dysregulated in newborn Jag1Ndr/Ndr mice, with aberrations in cholangiocyte polarity, but these defects improved in adult mice. Jag1Ndr/Ndr liver organoids collapsed in culture, indicating structural instability. Whole-transcriptome sequence analyses of liver tissues from mice and patients with Alagille syndrome identified dysregulated genes encoding proteins enriched at the apical side of cholangiocytes, including CFTR and SLC5A1, as well as reduced expression of IGF1. Exposure of Notch-expressing cells to JAG1Ndr, compared with JAG1, led to hypomorphic Notch signaling, based on transcriptome analysis. JAG1-expressing cells, but not JAG1Ndr-expressing cells, bound soluble Notch1 extracellular domain, quantified by flow cytometry. However, JAG1 and JAG1Ndr cells each bound NOTCH2, and signaling from NOTCH2 signaling was reduced but not completely inhibited, in response to JAG1Ndr compared with JAG1. CONCLUSIONS: In mice, expression of a missense mutant of Jag1 (Jag1Ndr) disrupts bile duct development and recapitulates Alagille syndrome phenotypes in heart, eye, and craniofacial dysmorphology. JAG1Ndr does not bind NOTCH1, but binds NOTCH2, and elicits hypomorphic signaling. This mouse model can be used to study other features of Alagille syndrome and organ development.

A liver-humanized mouse model of carbamoyl phosphate synthetase 1-deficiency.

A liver-humanized mouse model for CPS1-deficiency was generated by the high-level repopulation of the mouse liver with CPS1-deficient human hepatocytes. When compared with mice that are highly repopulated with CPS1-proficient human hepatocytes, mice that are repopulated with CPS1-deficient human hepatocytes exhibited characteristic symptoms of human CPS1 deficiency including an 80% reduction in CPS1 metabolic activity, delayed clearance of an ammonium chloride infusion, elevated glutamine and glutamate levels, and impaired metabolism of [15 N]ammonium chloride into urea, with no other obvious phenotypic differences. Because most metabolic liver diseases result from mutations that alter critical pathways in hepatocytes, a model that incorporates actual disease-affected, mutant human hepatocytes is useful for the investigation of the molecular, biochemical, and phenotypic differences induced by that mutation. The model is also expected to be useful for investigations of modified RNA, gene, and cellular and small molecule therapies for CPS1-deficiency. Liver-humanized models for this and other monogenic liver diseases afford the ability to assess the therapy on actual disease-affected human hepatocytes, in vivo, for long periods of time and will provide data that are highly relevant for investigations of the safety and efficacy of gene-editing technologies directed to human hepatocytes and the translation of gene-editing technology to the clinic.

Characterisation of the NRF2 transcriptional network and its response to chemical insult in primary human hepatocytes: implications for prediction of drug-induced liver injury.

The transcription factor NRF2, governed by its repressor KEAP1, protects cells against oxidative stress. There is interest in modelling the NRF2 response to improve the prediction of clinical toxicities such as drug-induced liver injury (DILI). However, very little is known about the makeup of the NRF2 transcriptional network and its response to chemical perturbation in primary human hepatocytes (PHH), which are often used as a translational model for investigating DILI. Here, microarray analysis identified 108 transcripts (including several putative novel NRF2-regulated genes) that were both downregulated by siRNA targeting NRF2 and upregulated by siRNA targeting KEAP1 in PHH. Applying weighted gene co-expression network analysis (WGCNA) to transcriptomic data from the Open TG-GATES toxicogenomics repository (representing PHH exposed to 158 compounds) revealed four co-expressed gene sets or 'modules' enriched for these and other NRF2-associated genes. By classifying the 158 TG-GATES compounds based on published evidence, and employing the four modules as network perturbation metrics, we found that the activation of NRF2 is a very good indicator of the intrinsic biochemical reactivity of a compound (i.e. its propensity to cause direct chemical stress), with relatively high sensitivity, specificity, accuracy and positive/negative predictive values. We also found that NRF2 activation has lower sensitivity for the prediction of clinical DILI risk, although relatively high specificity and positive predictive values indicate that false positive detection rates are likely to be low in this setting. Underpinned by our comprehensive analysis, activation of the NRF2 network is one of several mechanism-based components that can be incorporated into holistic systems toxicology models to improve mechanistic understanding and preclinical prediction of DILI in man.

Composition and functionality of the intrahepatic innate lymphoid cell-compartment in human nonfibrotic and fibrotic livers.

Human innate lymphoid cells have been described to exist in different organs, with functional deregulation of these cells contributing to several disease states. Here, we performed the first detailed characterization of the phenotype, tissue-residency properties, and functionality of ILC1s, ILC2s, and ILC3s in the human adult and fetal liver. In addition, we investigated changes in the ILC compartment in liver fibrosis. A unique composition of tissue-resident ILCs was observed in nonfibrotic livers as compared with that in mucosal tissues, with NKp44- ILC3s accounting for the majority of total intrahepatic ILCs. The frequency of ILC2s, representing a small fraction of ILCs in nonfibrotic livers, increased in liver fibrosis and correlated directly with the severity of the disease. Notably, intrahepatic ILC2s secreted the profibrotic cytokine IL-13 when exposed to IL-33 and thymic stromal lymphopoetin (TSLP); these cytokines were produced by hepatocytes, hepatic stellate cells (HSCs), and Kupffer cells in response to TLR-3 stimulation. In summary, the present results provide the first detailed characterization of intrahepatic ILCs in human adult and fetal liver. The results indicate a role for ILC2s in human liver fibrosis, implying that targeting ILC2s might be a novel therapeutic strategy for its treatment.

Serial Assessment of Growth Factors Associated with Liver Regeneration in Patients Operated with Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy.

BACKGROUND: There is limited knowledge about the mechanisms behind the unparalleled growth of the future liver remnant (FLR) linked to associating liver partition and portal vein ligation for staged hepatectomy (ALPPS). In this study, liver regenerative markers were examined in patients subjected to ALPPS. METHODS: Ten patients with colorectal liver metastases treated with neoadjuvant chemotherapy and ALPPS were included. Plasma was sampled at 6 time points and biopsies from both liver lobes were collected at both stages of ALPPS. The levels of interleukin (IL)-6, hepatocyte growth factor (HGF), tumor necrosis factor-α, epidermal growth factor, and vascular endothelial growth factor in plasma were measured at each time point. Expression of mRNA for markers of proliferation and apoptosis was studied in the biopsies from both liver lobes taken at both stages. RESULTS: ALPPS resulted in a peak of IL-6 after stage 1 (p = 0.004), which decreased rapidly and did not increase again after stage 2. HGF also increased after stage 1 (p = 0.048), and the HGF levels correlated significantly with the degree of growth of the FLR before stage 2 (p = 0.02, r2 = 0.47). There was a correlation between peak levels of IL-6 and HGF (p = 0.03, r2 = 0.84). CONCLUSIONS: IL-6 and HGF seem to be early mediators of hypertrophy after stage 1 in the ALPPS procedure. The peak HGF plasma level correlates with the degree of FLR growth in patients subjected to ALPPS.

GPS2-dependent corepressor/SUMO pathways govern anti-inflammatory actions of LRH-1 and LXRbeta in the hepatic acute phase response.

The orphan receptor LRH-1 and the oxysterol receptors LXRalpha and LXRbeta are established transcriptional regulators of lipid metabolism that appear to control inflammatory processes. Here, we investigate the anti-inflammatory actions of these nuclear receptors in the hepatic acute phase response (APR). We report that selective synthetic agonists induce SUMOylation-dependent recruitment of either LRH-1 or LXR to hepatic APR promoters and prevent the clearance of the N-CoR corepressor complex upon cytokine stimulation. Investigations of the APR in vivo, using LXR knockout mice, indicate that the anti-inflammatory actions of LXR agonists are triggered selectively by the LXRbeta subtype. We further find that hepatic APR responses in small ubiquitin-like modifier-1 (SUMO-1) knockout mice are increased, which is due in part to diminished LRH-1 action at APR promoters. Finally, we provide evidence that the metabolically important coregulator GPS2 functions as a hitherto unrecognized transrepression mediator of interactions between SUMOylated nuclear receptors and the N-CoR corepressor complex. Our study extends the knowledge of anti-inflammatory mechanisms and pathways directed by metabolic nuclear receptor-corepressor networks to the control of the hepatic APR, and implies alternative pharmacological strategies for the treatment of human metabolic diseases associated with inflammation.

Massive rearrangements of cellular MicroRNA signatures are key drivers of hepatocyte dedifferentiation.

Hepatocytes are dynamic cells that, upon injury, can alternate between nondividing differentiated and dedifferentiated proliferating states in vivo. However, in two-dimensional cultures, primary human hepatocytes (PHHs) rapidly dedifferentiate, resulting in loss of hepatic functions that significantly limits their usefulness as an in vitro model of liver biology, liver diseases, as well as drug metabolism and toxicity. Thus, understanding the underlying mechanisms and stalling of the dedifferentiation process would be highly beneficial to establish more-accurate and relevant long-term in vitro hepatocyte models. Here, we present comprehensive analyses of whole proteome and transcriptome dynamics during the initiation of dedifferentiation during the first 24 hours of culture. We report that early major rearrangements of the noncoding transcriptome, hallmarked by increased expression of small nucleolar RNAs, long noncoding RNAs, microRNAs (miRNAs), and ribosomal genes, precede most changes in coding genes during dedifferentiation of PHHs, and we speculated that these modulations could drive the hepatic dedifferentiation process. To functionally test this hypothesis, we globally inhibited the miRNA machinery using two established chemically distinct compounds, acriflavine and poly-l-lysine. These inhibition experiments resulted in a significantly impaired miRNA response and, most important, in a pronounced reduction in the down-regulation of hepatic genes with importance for liver function. Thus, we provide strong evidence for the importance of noncoding RNAs, in particular, miRNAs, in hepatic dedifferentiation, which can aid the development of more-efficient differentiation protocols for stem-cell-derived hepatocytes and broaden our understanding of the dynamic properties of hepatocytes with respect to liver regeneration. CONCLUSION: miRNAs are important drivers of hepatic dedifferentiation, and our results provide valuable information regarding the mechanisms behind liver regeneration and possibilities to inhibit dedifferentiation in vitro. (Hepatology 2016;64:1743-1756).

Cutting edge: identification and characterization of human intrahepatic CD49a+ NK cells.

Although NK cells are considered innate, recent studies in mice revealed the existence of a unique lineage of hepatic CD49a(+)DX5(-) NK cells with adaptive-like features. Development of this NK cell lineage is, in contrast to conventional NK cells, dependent on T-bet but not Eomes. In this study, we describe the identification of a T-bet(+)Eomes(-)CD49a(+) NK cell subset readily detectable in the human liver, but not in afferent or efferent hepatic venous or peripheral blood. Human intrahepatic CD49a(+) NK cells express killer cell Ig-like receptor and NKG2C, indicative of having undergone clonal-like expansion, are CD56(bright), and express low levels of CD16, CD57, and perforin. After stimulation, CD49a(+) NK cells express high levels of inflammatory cytokines but degranulate poorly. CD49a(+) NK cells retain their phenotype after expansion in long-term in vitro cultures. These results demonstrate the presence of a likely human counterpart of mouse intrahepatic NK cells with adaptive-like features.

Bile acid signaling pathways increase stability of Small Heterodimer Partner (SHP) by inhibiting ubiquitin-proteasomal degradation.

Small Heterodimer Partner (SHP) inhibits activities of numerous transcription factors involved in diverse biological pathways. As an important metabolic regulator, SHP plays a key role in maintaining cholesterol and bile acid homeostasis by inhibiting cholesterol conversion to bile acids. While SHP gene induction by increased bile acids is well established, whether SHP activity is also modulated remains unknown. Here, we report surprising findings that SHP is a rapidly degraded protein via the ubiquitin-proteasomal pathway and that bile acids or bile acid-induced intestinal fibroblast growth factor 19 (FGF19) increases stability of hepatic SHP by inhibiting proteasomal degradation in an extracellular signal-regulated kinase (ERK)-dependent manner. SHP was ubiquitinated at Lys122 and Lys123, and mutation of these sites altered its stability and repression activity. Tandem mass spectrometry revealed that upon bile acid treatment, SHP was phosphorylated at Ser26, within an ERK motif in SHP, and mutation of this site dramatically abolished SHP stability. Surprisingly, SHP stability was abnormally elevated in ob/ob mice and diet-induced obese mice. These results demonstrate an important role for regulation of SHP stability in bile acid signaling in normal conditions, and that abnormal stabilization of SHP may be associated with metabolic disorders, including obesity and diabetes.

Changes in gluconeogenesis and intracellular lipid accumulation characterize uremic human hepatocytes ex vivo.

It is well known that reduced glomerular filtration rate (GFR) leads to an increased risk of dyslipidemia, insulin resistance, and cardiovascular mortality. The liver is a central organ for metabolism, but its function in the uremic setting is still poorly characterized. We used human primary hepatocytes isolated from livers of nine donors with normal renal function to investigate perturbations in key metabolic pathways following exposure to uremic (n = 8) or healthy (n = 8) sera, and to serum-free control medium. Both uremic and healthy elicited consistent responses from hepatocytes from multiple donors and compared with serum-free control. However, at physiological insulin concentrations, uremic cells accumulated 56% more intracellular lipids. Also, when comparing uremic with healthy medium after culture, it contained more very-low-density lipoprotein-triglyceride and glucose. These changes were accompanied by decreased phosphorylation of AktS473 mRNA levels of key regulators of gluconeogenesis in uremic sera-treated hepatocytes such as phosphoenolpyruvate carboxykinase 1 and glucose 6-phosphate were elevated. We also found increased expression of 11ß-hydroxysteroid dehydrogenase mRNA in uremic cells, along with high phosphorylation of downstream p53 and phospholipase C-γ1Y783 Thus our ex vivo data suggest that the uremic hepatocytes rapidly develop a glycogenic and lipogenic condition accompanied by perturbations in a large number of signaling networks.