mARC1 in MASLD: Modulation of lipid accumulation in human hepatocytes and adipocytes.

BACKGROUND: Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models. METHODS AND RESULTS: We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models. CONCLUSIONS: Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.

Alpha-fetoprotein: Past, present, and future.

Alpha-fetoprotein (AFP) is a glycoprotein that plays an important role in immune regulation with critical involvement in early human development and maintaining the immune balance during pregnancy. Postfetal development, the regulatory mechanisms controlling AFP undergo a shift and AFP gene transcription is suppressed. Instead, these enhancers refocus their activity to maintain albumin gene transcription throughout adulthood. During the postnatal period, AFP expression can increase in the setting of hepatocyte injury, regeneration, and malignant transformation. It is the first oncoprotein discovered and is routinely used as part of a screening strategy for HCC. AFP has been shown to be a powerful prognostic biomarker, and multiple HCC prognosis models confirmed the independent prognostic utility of AFP. AFP is also a useful predictive biomarker for monitoring the treatment response of HCC. In addition to its role as a biomarker, AFP plays important roles in immune modulation to promote tumorigenesis and thus has been investigated as a therapeutic target in HCC. In this review article, we aim to provide an overview of AFP, encompassing the discovery, biological role, and utility as an HCC biomarker in combination with other biomarkers and how it impacts clinical practice and future direction.

Interferon-α stimulates DExH-box helicase 58 to prevent hepatocyte ferroptosis.

BACKGROUND: Liver ischemia/reperfusion (I/R) injury is usually caused by hepatic inflow occlusion during liver surgery, and is frequently observed during war wounds and trauma. Hepatocyte ferroptosis plays a critical role in liver I/R injury, however, it remains unclear whether this process is controlled or regulated by members of the DEAD/DExH-box helicase (DDX/DHX) family. METHODS: The expression of DDX/DHX family members during liver I/R injury was screened using transcriptome analysis. Hepatocyte-specific Dhx58 knockout mice were constructed, and a partial liver I/R operation was performed. Single-cell RNA sequencing (scRNA-seq) in the liver post I/R suggested enhanced ferroptosis by Dhx58hep-/-. The mRNAs and proteins associated with DExH-box helicase 58 (DHX58) were screened using RNA immunoprecipitation-sequencing (RIP-seq) and IP-mass spectrometry (IP-MS). RESULTS: Excessive production of reactive oxygen species (ROS) decreased the expression of the IFN-stimulated gene Dhx58 in hepatocytes and promoted hepatic ferroptosis, while treatment using IFN-α increased DHX58 expression and prevented ferroptosis during liver I/R injury. Mechanistically, DHX58 with RNA-binding activity constitutively associates with the mRNA of glutathione peroxidase 4 (GPX4), a central ferroptosis suppressor, and recruits the m6A reader YT521-B homology domain containing 2 (YTHDC2) to promote the translation of Gpx4 mRNA in an m6A-dependent manner, thus enhancing GPX4 protein levels and preventing hepatic ferroptosis. CONCLUSIONS: This study provides mechanistic evidence that IFN-α stimulates DHX58 to promote the translation of m6A-modified Gpx4 mRNA, suggesting the potential clinical application of IFN-α in the prevention of hepatic ferroptosis during liver I/R injury.

Traditional and emerging strategies using hepatocytes for pancreatic regenerative medicine.

Although pancreas and islet cell transplantation are the only ways to prevent the late complications of insulin-dependent diabetes, a shortage of donors is a major obstacle to tissue and organ transplantation. Stem cell therapy is an effective treatment for diabetes and other pancreatic-related diseases, which can be achieved by inducing their differentiation into insulin-secreting cells. The liver is considered an ideal source of pancreatic cells due to its similar developmental origin and strong regenerative ability as the pancreas. This article reviews the traditional and emerging strategies using hepatocytes for pancreatic regenerative medicine and evaluates their advantages and challenges. Gene reprogramming and chemical reprogramming technologies are traditional strategies with potential to improve the efficiency and specificity of cell reprogramming and promote the transformation of hepatocytes into islet cells. At the same time, organoid technology, as an emerging strategy, has received extensive attention. Biomaterials provide a three-dimensional culture microenvironment for cells, which helps improve cell survival and differentiation efficiency. In addition, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology has brought new opportunities and challenges to the development of organoid technology.

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.

Acute exposure to polystyrene nanoparticles promotes liver injury by inducing mitochondrial ROS-dependent necroptosis and augmenting macrophage-hepatocyte crosstalk.

BACKGROUND: The global use of plastic materials has undergone rapid expansion, resulting in the substantial generation of degraded and synthetic microplastics and nanoplastics (MNPs), which have the potential to impose significant environmental burdens and cause harmful effects on living organisms. Despite this, the detrimental impacts of MNPs exposure towards host cells and tissues have not been thoroughly characterized. RESULTS: In the present study, we have elucidated a previously unidentified hepatotoxic effect of 20 nm synthetic polystyrene nanoparticles (PSNPs), rather than larger PS beads, by selectively inducing necroptosis in macrophages. Mechanistically, 20 nm PSNPs were rapidly internalized by macrophages and accumulated in the mitochondria, where they disrupted mitochondrial integrity, leading to heightened production of mitochondrial reactive oxygen species (mtROS). This elevated mtROS generation essentially triggered necroptosis in macrophages, resulting in enhanced crosstalk with hepatocytes, ultimately leading to hepatocyte damage. Additionally, it was demonstrated that PSNPs induced necroptosis and promoted acute liver injury in mice. This harmful effect was significantly mitigated by the administration of a necroptosis inhibitor or systemic depletion of macrophages prior to PSNPs injection. CONCLUSION: Collectively, our study suggests a profound toxicity of environmental PSNP exposure by triggering macrophage necroptosis, which in turn induces hepatotoxicity via intercellular crosstalk between macrophages and hepatocytes in the hepatic microenvironment.

In Silico and Chromatographic Methods for Analysis of Biotransformation of Prospective Neuroprotective Pyrrole-Based Hydrazone in Isolated Rat Hepatocytes.

In the current study, chromatographic and in silico techniques were applied to investigate the biotransformation of ethyl 5-(4-bromophenyl)-1-(2-(2-(2-hydroxybenzylidene) hydrazinyl)-2-oxoethyl)-2-methyl-1H-pyrrole-3-carboxylate (11b) in hepatocytic media. The initial chromatographic procedure was based on the employment of the conventional octadecyl stationary phase method for estimation of the chemical stability. Subsequently, a novel and rapid chromatographic approach based on a phenyl-hexyl column was developed, aiming to separate the possible metabolites. Both methods were performed on a Dionex 3000 ThermoScientific (ACM 2, Sofia, Bulgaria) device equipped with a diode array detector set up at 272 and 279 nm for analytes detection. An acetonitrile: phosphate buffer of pH 3.5: methanol (17:30:53 v/v/v) was eluted isocratically as a mobile phase with a 1 mL/min flow rate. A preliminary purification from the biological media was achieved by protein precipitation with methanol. A validation procedure was carried out, where the method was found to correspond to all ICH (Q2) and M10 set criteria. Additionally, an in silico-based approach with the online server BioTransformer 3.0 was applied in an attempt to predict the possible metabolites of the title compound 11b. It was hypothesized that four CYP450 isoforms (1A2, 2C9, 3A4, and 2C8) were involved in the phase I metabolism, resulting in the formation of 12 metabolites. Moreover, docking studies were conducted to evaluate the formation of stable complexes between 11b and the aforementioned isoforms. The obtained data indicated three metabolites as the most probable products, two of which (M9_11b and M10_11b) were synthesized by a classical approach for verification. Finally, liquid chromatography with a mass detector was implemented for comprehensive and summarized analysis, and the obtained results revealed that the metabolism of the 11b proceeds possibly with the formation of glucuronide and glycine conjugate of M11_11b.

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.

Protective hepatocyte signals restrain liver fibrosis in metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects nearly 40% of the global adult population and may progress to metabolic dysfunction-associated steatohepatitis (MASH), and MASH-associated liver fibrosis and cirrhosis. Despite numerous studies unraveling the mechanism of hepatic fibrogenesis, there are still no approved antifibrotic therapies. The development of MASLD and liver fibrosis results from complex cell-cell interactions that often initiate within hepatocytes but remain incompletely understood. In this issue of the JCI, Yan and colleagues describe an ATF3/HES1/CEBPA/OPN pathway that links hepatocyte signals to fibrogenic activation of hepatic stellate cells and may provide new perspectives on therapeutic options for MASLD-induced liver fibrosis.

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.

Hepatocyte regeneration is driven by embryo-like DNA methylation reprogramming.

As a result of partial hepatectomy, the remaining liver tissue undergoes a process of renewed proliferation that leads to rapid regeneration of the liver. By following the early stages of this process, we observed dramatic programmed changes in the DNA methylation profile, characterized by both de novo and demethylation events, with a subsequent return to the original adult pattern as the liver matures. Strikingly, these transient alterations partially mimic the DNA methylation state of embryonic hepatoblasts (E16.5), indicating that hepatocytes actually undergo epigenetic dedifferentiation. Furthermore, Tet2/Tet3-deletion experiments demonstrated that these changes in methylation are necessary for carrying out basic embryonic functions, such as proliferation, a key step in liver regeneration. This implies that unlike tissue-specific regulatory regions that remain demethylated in the adult, early embryonic genes are programmed to first undergo demethylation, followed by remethylation as development proceeds. The identification of this built-in system may open targeting opportunities for regenerative medicine.

Chlorantraniliprole induces mitophagy, ferroptosis, and cytokine homeostasis imbalance in grass carp (Ctenopharyngodon idella) hepatocytes via the mtROS-mitochondrial fission/fusion axis.

Chlorantraniliprole (CAP) is a bis-amide pesticide used for pest control mainly in agricultural production activities and rice-fish co-culture systems. CAP residues cause liver damage in non-target organism freshwater fish. However, it is unclear whether CAP-exposure-induced liver injury in fish is associated with mitochondrial dysfunction-mediated mitophagy, ferroptosis, and cytokines. Therefore, we established grass carp hepatocyte models exposed to different concentrations of CAP (20, 40, and 80 µM) in vitro. MitoSOX probe, JC-1 staining, immunofluorescence double staining, Fe2+ staining, lipid peroxidation staining, qRT-PCR, and Western blot were used to verify the physiological regulatory mechanism of CAP induced liver injury. In the present study, the CAP-treated groups exhibited down-regulation of antioxidant-related enzyme activities and accumulation of peroxides. CAP treatment induced an increase in mitochondrial reactive oxygen species (mtROS) levels and altered expression of mitochondrial fission/fusion (Drp1, Fis1, Mfn1, Mfn2, and Opa1) genes in grass carp hepatocytes. In addition, mitophagy (Parkin, Pink1, p62, LC3II/I, and Beclin-1), ferroptosis (GPX4, COX2, ACSL4, FTH, and NCOA4), and cytokine (IFN-γ, IL-18, IL-17, IL-6, IL-10, IL-1ß, IL-2, and TNF-α)-related gene expression was significantly altered. Collectively, these findings suggest that CAP exposure drives mitophagy activation, ferroptosis occurrence, and cytokine homeostasis imbalance in grass carp hepatocytes by triggering mitochondrial dysfunction mediated by the mtROS-mitochondrial fission/fusion axis. This study partly explained the physiological regulation mechanism of grass carp hepatocyte injury induced by insecticide CAP from the physiological and biochemical point of view and provided a basis for evaluating the safety of CAP environmental residues to non-target organisms.

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.

Phosphoglycerate mutase 5 exacerbates liver ischemia-reperfusion injury by activating mitochondrial fission.

Although the death of hepatocytes is a crucial trigger of liver ischemia-reperfusion (I/R) injury, the regulation of liver I/R-induced hepatocyte death is still poorly understood. Phosphoglycerate mutase 5 (PGAM5), a mitochondrial Serine/Threonine protein phosphatase, regulates mitochondrial dynamics and is involved in the process of both apoptosis and necrotic. However, it is still unclear what role PGAM5 plays in the death of hepatocytes induced by I/R. Using a PGAM5-silence mice model, we investigated the role of PGAM5 in liver I/R injury and its relevant molecular mechanisms. Our data showed that PGAM5 was highly expressed in mice with liver I/R injury. Silence of PGAM5 could decrease I/R-induced hepatocyte death in mice. In subcellular levels, the silence of PGAM5 could restore mitochondrial membrane potential, increase mitochondrial DNA copy number and transcription levels, inhibit ROS generation, and prevent I/R-induced opening of abnormal mPTP. As for the molecular mechanisms, we indicated that the silence of PGAM5 could inhibit Drp1(S616) phosphorylation, leading to a partial reduction of mitochondrial fission. In addition, Mdivi-1 could inhibit mitochondrial fission, decrease hepatocyte death, and attenuate liver I/R injury in mice. In conclusion, our data reveal the molecular mechanism of PGAM5 in driving hepatocyte death through activating mitochondrial fission in liver I/R injury.

Perfluorooctanoic acid inhibits cell proliferation through mitochondrial damage.

Grown evidence has shown that the liver and reproductive organs were the main target organs of perfluorooctanoic acid (PFOA). Herein, we studied a toxic mechanism of PFOA using HeLa Chang liver epithelial cells. When incubated with PFOA for 24 h or 48 h, cell proliferation was inhibited in a concentration- and time-dependent fashion, but interestingly, the feature of dead cells was not notable. Mitochondrial volume was increased with concentration and time, whereas the mitochondrial membrane potential and produced ATP amounts were significantly reduced. Autophagosome-like vacuoles and contraction of the mitochondrial inner membrane were observed in PFOA-treated cells. The expression of acetyl CoA carboxylase (ACC) and p-ACC proteins rapidly decreased, and that of mitochondrial dynamics-related proteins increased. The expression of solute carrier family 7 genes, ChaC glutathione-specific gamma-glutamylcyclotransferase 1, and 5S ribosomal RNA gene was up-regulated the most in cells exposed to PFOA for 24 h, and the KEGG pathway analysis revealed that PFOA the most affected metabolic pathways and olfactory transduction. More importantly, PPAR alpha, fatty acid binding protein 1, and CYP450 family 1 subfamily A member 1 were identified as the target proteins for binding between PFOA and cells. Taken together, we suggest that disruption of mitochondrial integrity and function may contribute closely to PFOA-induced cell proliferation inhibition.

Si-Wu-Tang attenuates hepatocyte PANoptosis and M1 polarization of macrophages in non-alcoholic fatty liver disease by influencing the intercellular transfer of mtDNA.

ETHNOPHARMACOLOGICAL RELEVANCE: Non-alcoholic fatty liver disease (NAFLD) represents a burgeoning challenge for public health with potential progression to malignant liver diseases. PANoptosis, an avant-garde conceptualization of cell deaths, is closely associated with mitochondrial damage and linked to multiple liver disorders. Si-Wu-Tang (SWT), a traditional Chinese herbal prescription renowned for regulating blood-related disorders and ameliorating gynecological and hepatic diseases, has been demonstrated to alleviate liver fibrosis by regulating bile acid metabolism and immune responses. AIM OF THE STUDY: However, the mechanisms by which mtDNA is released from PANoptotic hepatocytes, triggering macrophage activation and hepatitis and whether this process can be reversed by SWT remain unclear. MATERIALS AND METHODS: Here, sophisticated RNA-sequencing complemented by molecular approaches were applied to explore the underlying mechanism of SWT against NAFLD in methionine/choline-deficient diet (MCD)-induced mice and relative in vitro models. RESULTS: We revealed that SWT profoundly repaired mitochondrial dysfunction, blocked mitochondrial permeability transition and mtDNA released to the cytoplasm, subsequently reversing hepatocyte PANoptosis and macrophage polarization both in MCD-stimulated mice and in vitro. Mechanically, loaded lipids dramatically promoted the opening of mPTP and oligomerization of VDAC2 to orchestrate mtDNA release, which was combined with ZBP1 to promote hepatocyte PANoptosis and also taken by macrophages to trigger M1 polarization via the FSTL1 and PKM2 combination. SWT effectively blocked NOXA signaling and reversed all these detrimental outcomes. CONCLUSION: Our findings show that SWT protects against hepatitis-mediated hepatocyte PANoptosis and macrophage M1 polarization by influencing intrahepatic synthesis, release and intercellular transfer of mtDNA, suggesting a potential therapeutic strategy for ameliorating NAFLD.

HMGB2 Promotes De Novo Lipogenesis to Accelerate Hepatocyte Proliferation During Liver Regeneration.

Liver regeneration is a well-orchestrated compensatory process that is regulated by multiple factors. We recently reported the importance of the chromatin protein, a high-mobility group box 2 (HMGB2) in mouse liver regeneration. However, the molecular mechanism remains unclear. In this study, we aimed to study how HMGB2 regulates hepatocyte proliferation during liver regeneration. Seventy-percent partial hepatectomy (PHx) was performed in wild-type (WT) and HMGB2-knockout (KO) mice, and the liver tissues were used for microarray, immunohistochemistry, quantitative polymerase chain reaction (qPCR), and Western blotting analyses. In the WT mice, HMGB2-positive hepatocytes colocalized with cell proliferation markers. In the HMGB2-KO mice, hepatocyte proliferation was significantly decreased. Oil Red O staining revealed the transient accumulation of lipid droplets at 12-24 hr after PHx in the WT mouse livers. In contrast, decreased amount of lipid droplets were found in HMGB2-KO mouse livers, and it was preserved until 36 hr. The microarray, immunohistochemistry, and qPCR results demonstrated that the expression of lipid metabolism-related genes was significantly decreased in the HMGB2-KO mouse livers. The in vitro experiments demonstrated that a decrease in the amount of lipid droplets correlated with decreased cell proliferation activity in HMGB2-knockdown cells. HMGB2 promotes de novo lipogenesis to accelerate hepatocyte proliferation during liver regeneration.

Hepatic nutrient and hormone signaling to mTORC1 instructs the postnatal metabolic zonation of the liver.

The metabolic functions of the liver are spatially organized in a phenomenon called zonation, linked to the differential exposure of portal and central hepatocytes to nutrient-rich blood. The mTORC1 signaling pathway controls cellular metabolism in response to nutrients and insulin fluctuations. Here we show that simultaneous genetic activation of nutrient and hormone signaling to mTORC1 in hepatocytes results in impaired establishment of postnatal metabolic and zonal identity of hepatocytes. Mutant hepatocytes fail to upregulate postnatally the expression of Frizzled receptors 1 and 8, and show reduced Wnt/ß-catenin activation. This defect, alongside diminished paracrine Wnt2 ligand expression by endothelial cells, underlies impaired postnatal maturation. Impaired zonation is recapitulated in a model of constant supply of nutrients by parenteral nutrition to piglets. Our work shows the role of hepatocyte sensing of fluctuations in nutrients and hormones for triggering a latent metabolic zonation program.

Aminobenzotriazole inhibits and induces several key drug metabolizing enzymes complicating its utility as a pan CYP inhibitor for reaction phenotyping.

Aminobenzotriazole (ABT) is commonly used as a non-selective inhibitor of cytochrome P450 (CYP) enzymes to assign contributions of CYP versus non-CYP pathways to the metabolism of new chemical entities. Despite widespread use, a systematic review of the drug-drug interaction (DDI) potential for ABT has not been published nor have the implications for using it in plated hepatocyte models for low clearance reaction phenotyping. The goal being to investigate the utility of ABT as a pan-CYP inhibitor for reaction phenotyping of low clearance compounds by evaluating stability over the incubation period, inhibition potential against UGT and sulfotransferase enzymes, and interaction with nuclear receptors involved in the regulation of drug metabolizing enzymes and transporters. Induction potential for additional inhibitors used to ascribe fraction metabolism (fm ), pathway including erythromycin, ketoconazole, azamulin, atipamezole, ZY12201, and quinidine was also investigated. ABT significantly inhibited the clearance of a non-selective UGT substrate 4-methylumbelliferone, with several UGTs shown to be inhibited using selective probe substrates in human hepatocytes and rUGTs. The inhibitors screened in the induction assay were shown to induce enzymes regulated through Aryl Hydrocarbon Receptor, Constitutive Androstane Receptor, and Pregnane X Receptor. Lastly, a case study identifying the mechanisms of a clinical DDI between Palbociclib and ARV-471 is provided as an example of the potential consequences of using ABT to derive fm . This work demonstrates that ABT is not an ideal pan-CYP inhibitor for reaction phenotyping of low clearance compounds and establishes a workflow that can be used to enable robust characterization of other prospective inhibitors.

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.