Comparative analysis of SEC61A1 mutant R236C in two patient-derived cellular platforms.

SEC61A1 encodes a central protein of the mammalian translocon and dysfunction results in severe disease. Recently, mutation R236C was identified in patients having autosomal dominant polycystic liver disease (ADPLD). The molecular phenotype of R236C was assessed in two cellular platforms. Cells were immortalized by retroviral transduction of an oncogene (UCi) or reprogrammed to induced pluripotent stem cells (iPSC) that were differentiated to cholangiocyte progenitor-like cells (CPLC). UCi and CPLC were subjected to analyses of molecular pathways that were associated with development of disease. UCi displayed markers of epithelial cells, while CPLCs expressed typical markers of both cholangiocytes and hepatocytes. Cells encoding R236C showed a stable, continuous proliferation in both platforms, however growth rates were reduced as compared to wildtype control. Autophagy, cAMP synthesis, and secretion of important marker proteins were reduced in R236C-expressing cells. In addition, R236C induced increased calcium leakiness from the ER to the cytoplasm. Upon oxidative stress, R236C led to a high induction of apoptosis and necrosis. Although the grade of aberrant cellular functions differed between the two platforms, the molecular phenotype of R236C was shared suggesting that the mutation, regardless of the cell type, has a dominant impact on disease-associated pathways.

Differences in diacylglycerol acyltransferases expression patterns and regulation cause distinct hepatic triglyceride deposition in fish.

Triglyceride (TAG) deposition in the liver is associated with metabolic disorders. In lower vertebrate, the propensity to accumulate hepatic TAG varies widely among fish species. Diacylglycerol acyltransferases (DGAT1 and DGAT2) are major enzymes for TAG synthesis. Here we show that large yellow croaker (Larimichthys crocea) has significantly higher hepatic TAG level than that in rainbow trout (Oncorhynchus mykiss) fed with same diet. Hepatic expression of DGATs genes in croaker is markedly higher compared with trout under physiological condition. Meanwhile, DGAT1 and DGAT2 in both croaker and trout are required for TAG synthesis and lipid droplet formation in vitro. Furthermore, oleic acid treatment increases DGAT1 expression in croaker hepatocytes rather than in trout and has no significant difference in DGAT2 expression in two fish species. Finally, effects of various transcription factors on croaker and trout DGAT1 promoter are studied. We find that DGAT1 is a target gene of the transcription factor CREBH in croaker rather than in trout. Overall, hepatic expression and transcriptional regulation of DGATs display significant species differences between croaker and trout with distinct hepatic triglyceride deposition, which bring new perspectives on the use of fish models for studying hepatic TAG deposition.

Uridine 5'-Diphospho-glucuronosyltransferase 1A3 (UGT1A3) Prediction of Hepatic Clearance of Organic Anion Transporting Polypeptide 1B3 (OATP1B3) Substrate Telmisartan by Glucuronidation Using In Vitro-In Vivo Extrapolation (IVIVE).

BACKGROUND AND OBJECTIVE: The prediction of pharmacokinetic parameters for drugs metabolised by cytochrome P450 enzymes has been the subject of active research for many years, while the application of in vitro-in vivo extrapolation (IVIVE) techniques for non-cytochrome P450 enzymes has not been thoroughly evaluated. There is still no established quantitative method for predicting hepatic clearance of drugs metabolised by uridine 5'-diphospho-glucuronosyltransferases (UGTs), not to mention those which undergo hepatic uptake. The objective of the study was to predict the human hepatic clearance for telmisartan based on in vitro metabolic stability and hepatic uptake results. METHODS: Telmisartan was examined in liver systems, allowing to estimate intrinsic clearance (CLint, in vitro) based on the substrate disappearance rate with the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) technique. Obtained CLint, in vitro values were corrected for corresponding unbound fractions. Prediction of human hepatic clearance was made from scaled unbound CLint, in vitro data with the use of the well-stirred model, and finally referenced to the literature value of observed clearance in humans, allowing determination of the essential scaling factors. RESULTS: The in vitro scaled CLint, in vitro by UGT1A3 was assessed using three systems, human hepatocytes, liver microsomes, and recombinant enzymes. Obtained values were scaled and hepatic metabolism clearance was predicted, resulting in significant clearance underprediction. Utilization of the extended clearance concept (ECC) and hepatic uptake improved prediction of hepatic metabolism clearance. The scaling factors for hepatocytes, assessing the in vitro-in vivo difference, changed from sixfold difference to only twofold difference with the application of the ECC. CONCLUSIONS: The study showed that taking into consideration hepatic uptake of a drug allows us to obtain satisfactory scaling factors, hence enabling the prediction of in vivo hepatic glucuronidation from in vitro data.

Identification and validation of a novel predictive signature based on hepatocyte-specific genes in hepatocellular carcinoma by integrated analysis of single-cell and bulk RNA sequencing.

BACKGROUND: Hepatocellular carcinoma represents a significant global burden in terms of cancer-related mortality, posing a substantial risk to human health. Despite the availability of various treatment modalities, the overall survival rates for patients with hepatocellular carcinoma remain suboptimal. The objective of this study was to explore the potential of novel biomarkers and to establish a novel predictive signature utilizing multiple transcriptome profiles. METHODS: The GSE115469 and CNP0000650 cohorts were utilized for single cell analysis and gene identification. The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) datasets were utilized in the development and evaluation of a predictive signature. The expressions of hepatocyte-specific genes were further validated using the GSE135631 cohort. Furthermore, immune infiltration results, immunotherapy response prediction, somatic mutation frequency, tumor mutation burden, and anticancer drug sensitivity were analyzed based on various risk scores. Subsequently, functional enrichment analysis was performed on the differential genes identified in the risk model. Moreover, we investigated the expression of particular genes in chronic liver diseases utilizing datasets GSE135251 and GSE142530. RESULTS: Our findings revealed hepatocyte-specific genes (ADH4, LCAT) with notable alterations during cell maturation and differentiation, leading to the development of a novel predictive signature. The analysis demonstrated the efficacy of the model in predicting outcomes, as evidenced by higher risk scores and poorer prognoses in the high-risk group. Additionally, a nomogram was devised to forecast the survival rates of patients at 1, 3, and 5 years. Our study demonstrated that the predictive model may play a role in modulating the immune microenvironment and impacting the anti-tumor immune response in hepatocellular carcinoma. The high-risk group exhibited a higher frequency of mutations and was more likely to benefit from immunotherapy as a treatment option. Additionally, we confirmed that the downregulation of hepatocyte-specific genes may indicate the progression of hepatocellular carcinoma and aid in the early diagnosis of the disease. CONCLUSION: Our research findings indicate that ADH4 and LCAT are genes that undergo significant changes during the differentiation of hepatocytes into cancer cells. Additionally, we have created a unique predictive signature based on genes specific to hepatocytes.

Competitive adsorption of microRNA-532-3p by circular RNA SOD2 activates Thioredoxin Interacting Protein/NLR family pyrin domain containing 3 pathway and promotes pyroptosis of non-alcoholic fatty hepatocytes.

OBJECTIVE: There is a growing body of evidence indicating that pyroptosis, a programmed cell death mechanism, plays a crucial role in the exacerbation of inflammation and fibrosis in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Circular RNAs (circRNAs), functioning as vital regulators within NAFLD, have been shown to mediate the process of cell pyroptosis. This study aims to elucidate the roles and mechanisms of circRNAs in NAFLD. METHODS: Utilizing a high-fat diet (HFD)-induced rat model for in vivo experimentation and hepatocytes treated with palmitic acid (PA) for in vitro models, we identified circular RNA SOD2 (circSOD2) as our circRNA of interest through analysis with the circMine database. The expression levels of associated genes and pyroptosis-related proteins were determined using quantitative real-time polymerase chain reaction and Western blotting, alongside immunohistochemistry. Serum liver function markers, cellular inflammatory cytokines, malondialdehyde, lactate dehydrogenase levels, and mitochondrial membrane potential, were assessed using enzyme-linked immunosorbent assay, standard assay kits, or JC-1 staining. Flow cytometry was employed to detect pyroptotic cells, and lipid deposition in liver tissues was observed via Oil Red O staining. The interactions between miR-532-3p/circSOD2 and miR-532-3p/Thioredoxin Interacting Protein (TXNIP) were validated through dual-luciferase reporter assays and RNA immunoprecipitation experiments. RESULTS: Our findings demonstrate that, in both in vivo and in vitro NAFLD models, there was an upregulation of circSOD2 and TXNIP, alongside a downregulation of miR-532-3p. Mechanistically, miR-532-3p directly bound to the 3'-UTR of TXNIP, thereby mediating inflammation and cell pyroptosis through targeting the TXNIP/NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling pathway. circSOD2 directly interacted with miR-532-3p, relieving the suppression on the TXNIP/NLRP3 signaling pathway. Functionally, the knockdown of circSOD2 or TXNIP improved hepatocyte pyroptosis; the deletion of miR-532-3p reversed the effects of circSOD2 knockdown, and the deletion of TXNIP reversed the effects of circSOD2 overexpression. Furthermore, the knockdown of circSOD2 significantly mitigated the progression of NAFLD in vivo. CONCLUSION: circSOD2 competitively sponges miR-532-3p to activate the TXNIP/NLRP3 inflammasome signaling pathway, promoting pyroptosis in NAFLD.

Ex vivo gene editing and cell therapy for hereditary tyrosinemia type 1.

BACKGROUND: We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1. METHODS: We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice. RESULTS: We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice. CONCLUSIONS: The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.

Naringenin inhibits APAP-induced acute liver injury through activating PPARA-dependent signaling pathway.

Acute liver injury (ALI) refers to the damage to the liver cells of patients due to drugs, food, and diseases. In this work, we used a network pharmacology approach to analyze the relevant targets and pathways of the active ingredients in Citri Reticulatae Pericarpium (CRP) for the treatment of ALI and conducted systematic validation through in vivo and in vitro experiments. The network pharmacologic results predicted that naringenin (NIN) was the main active component of CRP in the treatment of ALI. GO functional annotation and KEGG pathway enrichment showed that its mechanism may be related to the regulation of PPARA signaling pathway, PPARG signaling pathway, AKT1 signaling pathway, MAPK3 signaling pathway and other signaling pathways. The results of in vivo experiments showed that (NIN) could reduce the liver lesions, liver adipose lesions, hepatocyte injury and apoptosis in mice with APAP-induced ALI, and reduce the oxidative stress damage of mouse liver cells and the inflammation-related factors to regulate ALI. In vitro experiments showed that NIN could inhibit the proliferation, oxidative stress and inflammation of APAP-induced LO2 cells, promote APAP-induced apoptosis of LO2 cells, and regulate the expression of apoptotic genes in acute liver injury. Further studies showed that NIN inhibited APAP-induced ALI mainly by regulating the PPARA-dependent signaling pathway. In conclusion, this study provides a preliminary theoretical basis for the screening of active compounds in CRP for the prevention and treatment of ALI.

Hepatocyte-specific CCAAT/enhancer binding protein α restricts liver fibrosis progression.

Metabolic dysfunction-associated steatohepatitis (MASH) - previously described as nonalcoholic steatohepatitis (NASH) - is a major driver of liver fibrosis in humans, while liver fibrosis is a key determinant of all-cause mortality in liver disease independent of MASH occurrence. CCAAT/enhancer binding protein α (CEBPA), as a versatile ligand-independent transcriptional factor, has an important function in myeloid cells, and is under clinical evaluation for cancer therapy. CEBPA is also expressed in hepatocytes and regulates glucolipid homeostasis; however, the role of hepatocyte-specific CEBPA in modulating liver fibrosis progression is largely unknown. Here, hepatic CEBPA expression was found to be decreased during MASH progression both in humans and mice, and hepatic CEBPA mRNA was negatively correlated with MASH fibrosis in the human liver. CebpaΔHep mice had markedly enhanced liver fibrosis induced by a high-fat, high-cholesterol, high-fructose diet or carbon tetrachloride. Temporal and spatial hepatocyte-specific CEBPA loss at the progressive stage of MASH in CebpaΔHep,ERT2 mice functionally promoted liver fibrosis. Mechanistically, hepatocyte CEBPA directly repressed Spp1 transactivation to reduce the secretion of osteopontin, a fibrogenesis inducer of hepatic stellate cells. Forced hepatocyte-specific CEBPA expression reduced MASH-associated liver fibrosis. These results demonstrate an important role for hepatocyte-specific CEBPA in liver fibrosis progression, and may help guide the therapeutic discoveries targeting hepatocyte CEBPA for the treatment of liver fibrosis.

Single-cell multiomics guided mechanistic understanding of Fontan-associated liver disease.

The Fontan operation is the current standard of care for single-ventricle congenital heart disease. Individuals with a Fontan circulation (FC) exhibit central venous hypertension and face life-threatening complications of hepatic fibrosis, known as Fontan-associated liver disease (FALD). The fundamental biology and mechanisms of FALD are little understood. Here, we generated a transcriptomic and epigenomic atlas of human FALD at single-cell resolution using multiomic snRNA-ATAC-seq. We found profound cell type-specific transcriptomic and epigenomic changes in FC livers. Central hepatocytes (cHep) exhibited the most substantial changes, featuring profound metabolic reprogramming. These cHep changes preceded substantial activation of hepatic stellate cells and liver fibrosis, suggesting cHep as a potential first "responder" in the pathogenesis of FALD. We also identified a network of ligand-receptor pairs that transmit signals from cHep to hepatic stellate cells, which may promote their activation and liver fibrosis. We further experimentally demonstrated that activins A and B promote fibrotic activation in vitro and identified mechanisms of activin A's transcriptional activation in FALD. Together, our single-cell transcriptomic and epigenomic atlas revealed mechanistic insights into the pathogenesis of FALD and may aid identification of potential therapeutic targets.

Liver cancer development driven by the AP-1/c-Jun~Fra-2 dimer through c-Myc.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. HCC incidence is on the rise, while treatment options remain limited. Thus, a better understanding of the molecular pathways involved in HCC development has become a priority to guide future therapies. While previous studies implicated the Activator Protein-1 (AP-1) (Fos/Jun) transcription factor family members c-Fos and c-Jun in HCC formation, the contribution of Fos-related antigens (Fra-) 1 and 2 is unknown. Here, we show that hepatocyte-restricted expression of a single chain c-Jun~Fra-2 protein, which functionally mimics the c-Jun/Fra-2 AP-1 dimer, results in spontaneous HCC formation in c-Jun~Fra-2hep mice. Several hallmarks of human HCC, such as cell cycle dysregulation and the expression of HCC markers are observed in liver tumors arising in c-Jun~Fra-2hep mice. Tumorigenesis occurs in the context of mild inflammation, low-grade fibrosis, and Pparγ-driven dyslipidemia. Subsequent analyses revealed increased expression of c-Myc, evidently under direct regulation by AP-1 through a conserved distal 3' enhancer. Importantly, c-Jun~Fra-2-induced tumors revert upon switching off transgene expression, suggesting oncogene addiction to the c-Jun~Fra-2 transgene. Tumors escaping reversion maintained c-Myc and c-Myc target gene expression, likely due to increased c-Fos. Interfering with c-Myc in established tumors using the Bromodomain and Extra-Terminal motif inhibitor JQ-1 diminished liver tumor growth in c-Jun~Fra-2 mutant mice. Thus, our data establish c-Jun~Fra-2hep mice as a model to study liver tumorigenesis and identify the c-Jun/Fra-2-Myc interaction as a potential target to improve HCC patient stratification and/or therapy.

Autonomous circadian rhythms in the human hepatocyte regulate hepatic drug metabolism and inflammatory responses.

Critical aspects of physiology and cell function exhibit self-sustained ~24-hour variations termed circadian rhythms. In the liver, circadian rhythms play fundamental roles in maintaining organ homeostasis. Here, we established and characterized an in vitro liver experimental system in which primary human hepatocytes display self-sustained oscillations. By generating gene expression profiles of these hepatocytes over time, we demonstrated that their transcriptional state is dynamic across 24 hours and identified a set of cycling genes with functions related to inflammation, drug metabolism, and energy homeostasis. We designed and tested a treatment protocol to minimize atorvastatin- and acetaminophen-induced hepatotoxicity. Last, we documented circadian-dependent induction of pro-inflammatory cytokines when triggered by LPS, IFN-ß, or Plasmodium infection in human hepatocytes. Collectively, our findings emphasize that the phase of the circadian cycle has a robust impact on the efficacy and toxicity of drugs, and we provide a test bed to study the timing and magnitude of inflammatory responses over the course of infection in human liver.

Increased susceptibility to ischemia causes exacerbated response to microinjuries in the cirrhotic liver.

Fractional laser ablation is a technique developed in dermatology to induce remodeling of skin scars by creating a dense pattern of microinjuries. Despite remarkable clinical results, this technique has yet to be tested for scars in other tissues. As a first step toward determining the suitability of this technique, we aimed to (1) characterize the response to microinjuries in the healthy and cirrhotic liver, and (2) determine the underlying cause for any differences in response. Healthy and cirrhotic rats were treated with a fractional laser then euthanized from 0 h up to 14 days after treatment. Differential expression was assessed using RNAseq with a difference-in-differences model. Spatial maps of tissue oxygenation were acquired with hyperspectral imaging and disruptions in blood supply were assessed with tomato lectin perfusion. Healthy rats showed little damage beyond the initial microinjury and healed completely by 7 days without scarring. In cirrhotic rats, hepatocytes surrounding microinjury sites died 4-6 h after ablation, resulting in enlarged and heterogeneous zones of cell death. Hepatocytes near blood vessels were spared, particularly near the highly vascularized septa. Gene sets related to ischemia and angiogenesis were enriched at 4 h. Laser-treated regions had reduced oxygen saturation and broadly disrupted perfusion of nodule microvasculature, which matched the zones of cell death. Our results demonstrate that the cirrhotic liver has an exacerbated response to microinjuries and increased susceptibility to ischemia from microvascular damage, likely related to the vascular derangements that occur during cirrhosis development. Modifications to the fractional laser tool, such as using a femtosecond laser or reducing the spot size, may be able to prevent large disruptions of perfusion and enable further development of a laser-induced microinjury treatment for cirrhosis.

Integrated transcriptomic and metabolomic analysis of the hepatotoxicity of dichloroacetonitrile.

Dichloroacetonitrile (DCAN), an emerged nitrogenous disinfection by-product (N-DBP) in drinking water, has garnered attention owing to its strong cytotoxicity, genotoxicity, and carcinogenicity. However, there are limited studies on its potential hepatotoxicity mechanisms. Understanding hepatotoxicity is essential in order to identify and assess the potential risks posed by environmental pollutants on liver health and to safeguard public health. Here, we investigated the viability, reactive oxygen species (ROS) levels, and cell cycle profile of DCAN-exposed HepG2 cells and analyzed the mechanism of DCAN-induced hepatotoxicity using both transcriptomic and metabolomic techniques. The study revealed that there was a decrease in cell viability, increase in ROS production, and increase in the number of cells in the G2/M phase with an increase in the concentration of DCAN. Omics analyses showed that DCAN exposure increased cellular ROS levels, leading to oxidative damage in hepatocytes, which further induced DNA damage, cell cycle arrest, and cell growth impairment. Thus, DCAN has significant toxic effects on hepatocytes. Integrated analysis of transcriptomics and metabolomics offers new insights into the mechanisms of DCAN-induced hepatoxicity.

Glycolysis is reduced in dengue virus 2 infected liver cells.

Infections with dengue virus (DENV) remain a worldwide public health problem. A number of bona fide cellular targets of DENV have been identified including liver cells. Despite the many lines of evidence confirming the involvement of hepatocytes during DENV infection, only a few studies have used proteomic analysis to understand the modulation of the cellular proteome occurring upon DENV infection. We utilized a 2D-gel electrophoresis analysis to identify proteins that were differentially regulated by DENV 2 infection of liver (Hep3B) cells at 12 h post infection (hpi) and at 48 hpi. The analysis identifies 4 proteins differentially expressed at 12 hpi, and 14 differentially regulated at 48 hpi. One candidate protein identified as downregulated at 48 hpi in the proteomic analysis (GAPDH) was validated in western blotting in Hep3B cells, and subsequently in induced pluripotent stem cell (iPSC) derived human hepatocytes. The reduced expression of GAPDH was coupled with an increase in NADH, and a significantly reduced NAD + /NADH ratio, strongly suggesting that glycolysis is down regulated in response to DENV 2 infection. Metformin, a well characterized drug used in the treatment of diabetes mellitus, is an inhibitor of hepatic gluconeogenesis was shown to reduce the level of DENV 2 infection and new virus production. Collectively these results show that although glycolysis is reduced, glucose is still required, possibly for use by the pentose phosphate pathway to generate nucleosides required for viral replication.

Multiple receptor tyrosine kinases regulate dengue infection of hepatocytes.

Introduction: Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. Methods: To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 291 kinases are regulating DENV infection. Results: Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of EPHB4, ERBB2, FGFR2, or IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Discussion: Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Adipocyte-derived exosomes from obstructive sleep apnoea rats aggravate MASLD by TCONS_00039830/miR-455-3p/Smad2 axis.

A correlation exists between obstructive sleep apnoea (OSA) and the severity of metabolic dysfunction-associated steatotic liver disease (MASLD), OSA can induce more severe MASLD. However, the underlying regulatory mechanism between the two is unclear. To this end, this study explored the role and possible molecular mechanisms of adipocyte-derived exosomes under OSA in aggravating MASLD. Through sequencing technology, miR-455-3p was identified as a co-differentially expressed miRNA between the MASLD + OSA and Control groups and between the MASLD + OSA and MASLD groups. Upregulation of TCONS-00039830 and Smad2 and downregulation of miR-455-3p in the MASLD and MASLD + OSA groups were validated in vivo and in vitro. TCONS-00039830, as a differentially expressed LncRNA in exosomes found in the sequencing results, transfection notably downregulated miR-455-3p and upregulated Smad2 in hepatocytes. TCONS_00039830 overexpression increased fat, triglyceride and cholesterol levels, while miR-455-3p overexpression decreased these levels. Furthermore, exosome administration promoted the accumulation of fat, triglyceride and cholesterol, upregulated TCONS_00039830 and Smad2, and downregulated miR-455-3p. Overexpression of miR-455-3p reversed the increased fat accumulation and upregulated TCONS_00039830 and Smad2. In conclusion, OSA-derived exosomes promoted hepatocyte steatosis by regulating TCONS_00039830/miR-455-3p/Smad2 axis, thereby aggravating liver damage in MASLD.

Anti-diabetic effect of anthocyanin cyanidin-3-O-glucoside: data from insulin resistant hepatocyte and diabetic mouse.

BACKGROUND: Anthocyanins are a group of natural products widely found in plants. They have been found to alleviate the disorders of glucose metabolism in type 2 diabetes mellitus (T2DM), while the underlying mechanisms remain unclear. METHODS: HepG2 and L02 cells were incubated with 0.2 mM PA and 30 mM glucose for 24 h to induce IR, and cells treated with 5 mM glucose were used as the control. C57BL/6 J male mice and db/db male mice were fed with a chow diet and gavaged with pure water or cyanidin-3-O-glucoside (C3G) solution (150 mg/kg/day) for 6 weeks. RESULTS: In this study, the anthocyanin C3G, extracted from red bayberry, was found to alleviate disorders of glucose metabolism, which resulted in increased insulin sensitivity in hepatocytes, and achieved by enhancing the glucose consumption as well as glycogen synthesis in insulin resistance (IR) hepatpcytes. Subsequently, the expression of key proteins involved in IR was detected by western blotting analysis. Protein tyrosine phosphatase-1B (PTP1B), a negative regulator of insulin signaling, could reduce cellular sensitivity to insulin by inhibiting the phosphorylation of insulin receptor substrate-2 (IRS-2). Results of this study showed that C3G inhibited the increase in PTP1B after high glucose and palmitic acid treatment. And this inhibition was accompanied by increased phosphorylation of IRS proteins. Furthermore, the effect of C3G on improving IR in vivo was validated by using a diabetic db/db mouse model. CONCLUSION: These findings demonstrated that C3G could alleviate IR in vitro and in vivo to increase insulin sensitivity, which may offer a new insight for regulating glucose metabolism during T2DM by using the natural dietary bioactive components. C3G promotes the phosphorylation of IRS-2 proteins by suppressing the expression of PTP1B, and then enhances the sensitivity of hepatocyte to insulin.

The micro-743a-3p-GSTM1 pathway is an endogenous protective mechanism against alcohol-related liver disease in mice.

BACKGROUND AND AIMS: Epidemiological evidence suggests that the phenotype of glutathione S-transferase mu 1 (GSTM1), a hepatic high-expressed phase II detoxification enzyme, is closely associated with the incidence of alcohol-related liver disease (ALD). However, whether and how hepatic GSTM1 determines the development of ALD is largely unclear. This study was designed to elucidate the role and potential mechanism(s) of hepatic GSTM1 in the pathological process of ALD. METHODS: GSTM1 was detected in the liver of various ALD mice models and cultured hepatocytes. Liver-specific GSTM1 or/and micro (miR)-743a-3p deficiency mice were generated by adenoassociated virus-8 delivered shRNA, respectively. The potential signal pathways involving in alcohol-regulated GSTM1 and GSTM1-associated ALD were explored via both genetic manipulation and pharmacological approaches. RESULTS: GSTM1 was significantly upregulated in both chronic alcohol-induced mice liver and ethanol-exposed murine primary hepatocytes. Alcohol-reduced miR-743a-3p directly contributed to the upregulation of GSTM1, since liver specific silencing miR-743a-3p enhanced GSTM1 and miR-743a-3p loss protected alcohol-induced liver dysfunctions, which was significantly blocked by GSTM1 knockdown. GSTM1 loss robustly aggravated alcohol-induced hepatic steatosis, oxidative stress, inflammation, and early fibrotic-like changes, which was associated with the activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK), and p38. GSTM1 antagonized ASK1 phosphorylation and its downstream JNK/p38 signaling pathway upon chronic alcohol consumption via binding with ASK1. ASK1 blockage significantly rescued hepatic GSTM1 loss-enhanced disorders in alcohol-fed mice liver. CONCLUSIONS: Chronic alcohol consumption-induced upregulation of GSTM1 in the liver provides a feedback protection against hepatic steatosis and liver injury by counteracting ASK1 activation. Down-regulation of miR-743a-3p improves alcohol intake-induced hepatic steatosis and liver injury via direct targeting on GSTM1. The miR-743a-3p-GSTM1 axis functions as an innate protective pathway to defend the early stage of ALD.

Cryopreserved cGMP-compliant human pluripotent stem cell-derived hepatic progenitors rescue mice from acute liver failure through rapid paracrine effects on liver cells.

BACKGROUND: Liver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. Considering their unique expansion capabilities and their potency to be driven toward a chosen cell fate, pluripotent stem cells are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failure and restore liver function. METHODS: Human PSC-derived immature hepatic progenitors (GStemHep) were generated using a new protocol with current good manufacturing practice compliant conditions from PSC amplification and hepatic differentiation to cell cryopreservation. The therapeutic potential of these cryopreserved cells was assessed in two clinically relevant models of acute liver failure, and the mode of action was studied by several analytical methods, including unbiased proteomic analyses. RESULTS: GStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor and do not express major histocompatibility complex. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute liver failure, both in immunodeficient and immunocompetent animals in the absence of immunosuppression. Therapeutic biological effects were observed as soon as 3 h post-cell transplantation with a reduction in serum transaminases and in liver necrosis. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation as well as a rapid cytoprotective effect with as a result a proteome reprograming of the host hepatocytes. The mode of action of GStemHep relie on the alleviation of inhibitory factors of liver regeneration, an increase in proliferation-promoting factors and a decrease in liver inflammation. CONCLUSIONS: We generated cryopreserved and current good manufacturing practice-compliant human pluripotent stem cell-derived immature hepatic progenitors that were highly effective in treating acute liver failure through rapid paracrine effects reprogramming endogenous hepatocytes. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in the absence of immunosuppression for human PSC-based regenerative medicine for acute liver failure.