Primary Murine Hepatocytes Research Articles

Shaoyao-Gancao Decoction, a famous Chinese medicine formula, protects against APAP-induced liver injury by promoting autophagy/mitophagy

BackgroundAcetaminophen (APAP)-induced hepatotoxicity is a major cause of acute liver failure (ALF), during which autophagy is triggered as a cellular defense mechanism. Shaoyao-Gancao Decoction (SGD), a traditional prescription in Chinese Medicine, is renowned for its therapeutic effects on liver diseases. However, the efficacy and mechanisms of SGD in treating APAP-induced liver injury remain underexplored. PurposeThis study aims to provide robust evidence regarding the protective effects of SGD against APAP overdose in vitro and in vivo, as well as to elucidate its hepatoprotective mechanisms and active components. Study designThe hepatoprotective mechanisms and active components of SGD were investigated through a combination of in vivo and in vitro experiments. MethodsThe protective effects of SGD on APAP-induced liver injury were assessed using a murine model and primary hepatocytes. RNA sequencing and subsequent experimental validations were conducted to uncover the underlying mechanisms of SGD's hepatoprotective actions. Comprehensive chemical profiling of SGD was performed using UHPLC-Q-Exactive Orbitrap HRMS to identify potential active ingredients. Immunohistochemistry, immunofluorescence, quantitative real-time PCR (qPCR), western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry were utilized to investigate the specific cellular changes in liver tissues and hepatocytes influenced by SGD. ResultsSGD was observed to mitigate APAP-induced mitochondrial damage, inflammation, and necrosis by promoting mitochondrial autophagy. The inhibition of autophagy negated the hepatoprotective effects of SGD. Additionally, a detailed characterization of SGD's chemical composition revealed that Licoisoflavone B, Liquiritin, Liquiritin apioside, Licorice saponin G2 and Paeoniflorin Sulfit were potentially critical compounds in the regulation of autophagy and mitophagy. ConclusionOur findings demonstrate that SGD promotes autophagy/mitophagy, which effectively mitigates APAP-induced hepatotoxicity, suggesting SGD's potential as a promising therapeutic agent for APAP-induced liver injury.

Genome Engineering of Primary and Pluripotent Stem Cell-Derived Hepatocytes for Modeling Liver Tumor Formation.

Genome editing has demonstrated its utility in generating isogenic cell-based disease models, enabling the precise introduction of genetic alterations into wild-type cells to mimic disease phenotypes and explore underlying mechanisms. However, its application in liver-related diseases has been limited by challenges in genetic modification of mature hepatocytes in a dish. Here, we conducted a systematic comparison of various methods for primary hepatocyte culture and gene delivery to achieve robust genome editing of hepatocytes ex vivo. Our efforts yielded editing efficiencies of up to 80% in primary murine hepatocytes cultured in monolayer and 20% in organoids. To model human hepatic tumorigenesis, we utilized hepatocytes differentiated from human pluripotent stem cells (hPSCs) as an alternative human hepatocyte source. We developed a series of cellular models by introducing various single or combined oncogenic alterations into hPSC-derived hepatocytes. Our findings demonstrated that distinct mutational patterns led to phenotypic variances, affecting both overgrowth and transcriptional profiles. Notably, we discovered that the PI3KCA E542K mutant, whether alone or in combination with exogenous c-MYC, significantly impaired hepatocyte functions and facilitated cancer metabolic reprogramming, highlighting the critical roles of these frequently mutated genes in driving liver neoplasia. In conclusion, our study demonstrates genome-engineered hepatocytes as valuable cellular models of hepatocarcinoma, providing insights into early tumorigenesis mechanisms.

Direct Ingestion of Oxidized Red Blood Cells (Efferocytosis) by Hepatocytes.

Both hepatic iron accumulation and hemolysis have been identified as independent prognostic factor in alcohol-related liver disease (ALD); however, the mechanisms still remain poorly understood. We here demonstrate that hepatocytes are able to directly ingest aged and ethanol-primed red blood cells (RBCs), a process termed efferocytosis. Efferocytosis of RBCs was directly studied in vitro and observed by live microscopy for real-time visualization. RBCs pretreated with either CuSO4 or ethanol following co-incubation with Huh7 cells and murine primary hepatocytes. Heme oxygenase-1 (HO-1) and other targets were measured by q-PCR. As shown by live microscopy, oxidized RBCs, but not intact RBCs, are rapidly ingested by both Huh7 cells and murine primary hepatocytes within 10 minutes. In some cases, more than 10 RBCs were seen within hepatocytes, surrounding the nucleus. RBC efferocytosis also rapidly induces HO1, its upstream regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and ferritin, indicating efficient heme degradation. Preliminary data further suggest that hepatocyte efferocytosis of oxidized RBCs is, at least in part, mediated by scavenging receptors such as ASGPR1. Of note, pretreatment of RBCs with ethanol but also heme and bilirubin also initiated efferocytosis. In a cohort of heavy human drinkers, a significant correlation of hepatic ASGPR1 with the heme degradation pathway was observed. We here demonstrate that hepatocytes can directly ingest and degrade oxidized RBCs through efferocytosis, a process that can be also triggered by ethanol, heme and bilirubin. Our findings are highly suggestive for a novel mechanism of hepatic iron overload in ALD patients.

Suppression of hepatic ChREBP⍺-CYP2C50 axis-driven fatty acid oxidation sensitizes mice to diet-induced MASLD/MASH

ObjectivesCompromised hepatic fatty acid oxidation (FAO) has been observed in human MASH patients and animal models of MASLD/MASH. It remains poorly understood how and when the hepatic FAO pathway is suppressed during the progression of MASLD towards MASH. Hepatic ChREBP⍺ is a classical lipogenic transcription factor that responds to the intake of dietary sugars. MethodsWe examined its role in regulating hepatocyte fatty acid oxidation (FAO) and the impact of hepatic Chrebpa deficiency on sensitivity to diet-induced MASLD/MASH in mice. ResultsWe discovered that hepatocyte ChREBP⍺ is both necessary and sufficient to maintain FAO in a cell-autonomous manner independently of its DNA-binding activity. Supplementation of synthetic PPAR⍺/δ agonist is sufficient to restore FAO in Chrebp−/− primary mouse hepatocytes. Hepatic ChREBP⍺ was decreased in mouse models of diet-induced MAFSLD/MASH and in patients with MASH. Hepatocyte-specific Chrebp⍺ knockout impaired FAO, aggravated liver steatosis and inflammation, leading to early-onset fibrosis in response to diet-induced MASH. Conversely, liver overexpression of ChREBP⍺-WT or its non-lipogenic mutant enhanced FAO, reduced lipid deposition, and alleviated liver injury, inflammation, and fibrosis. RNA-seq analysis identified the CYP450 epoxygenase (CYP2C50) pathway of arachidonic acid metabolism as a novel target of ChREBP⍺. Over-expression of CYP2C50 partially restores hepatic FAO in primary hepatocytes with Chrebp⍺ deficiency and attenuates preexisting MASH in the livers of hepatocyte-specific Chrebp⍺-deleted mice. ConclusionsOur findings support the protective role of hepatocyte ChREBPa against diet-induced MASLD/MASH in mouse models in part via promoting CYP2C50-driven FAO.

Abstract 158: The Role Of Ehbp1 In Hypercholesterolemia

Background: Despite aggressive lowering of cholesterol, conventional lipid-lowering therapies only modestly lower cardiovascular risk in the population. Therefore, identifying novel hypercholesterolemia-risk targets is of critical importance. Human genome-wide association studies (GWAS) have revealed that several single-nucleotide polymorphisms (SNPs) in EHBP1 , the gene that encodes EH domain binding protein 1 (EHBP1), an endocytic trafficking regulator, are associated with serum cholesterol. However, the mechanisms underlying this association and the role of EHBP1 in cholesterol metabolism are unknown. We hypothesize that EHBP1 is a novel hypercholesterolemia risk factor. The goal of this study is to explore the role of EHBP1 in hypercholesterolemia and its underlying mechanisms. Methods: Hepatocyte EHBP1 was silenced by intravenous injection (IV) of AAV8-H1-shEhbp1 in wild-type (WT), Apoe-/- and Ldlr-/- mice, followed by Western Diet feeding to induce hypercholesterolemia and atherosclerosis. Primary murine and human hepatocytes were isolated for in vitro mechanistic studies. We also leveraged publicly available human genetic studies and unbiased scRNA datasets to support our study. Results: Individuals with the EHBP1 rs10496099 T>C variant have decreased serum cholesterol and reduced liver EHBP1 expression. Consistent with the human data, our in vivo study with hypercholesterolemic mice shows that EHBP1 silencing in hepatocytes decreases plasma total and LDL/VLDL cholesterol. Our in vitro study with primary hepatocytes reveals that EHBP1 deficiency eliminates sortilin (SORT1)-mediated PCSK9 secretion and induces LDLR-mediated LDL uptake. Mechanistically, we found that EHBP1 silencing abolishes the homeostasis of the retromer complex, a protein complex that helps retrograde and stabilize sortilin. Conclusion: Hepatocyte EHBP1 promotes sortilin-mediated PCSK9 secretion by maintaining retromer homeostasis, leading to LDLR degradation and hypercholesterolemia. Our study elucidates a novel link between EHBP1 and other cholesterol-associated loci (e.g., SORT1 , PCSK9 , and LDLR) and provides new mechanistic insight into the role of EHBP1 in hepatic cholesterol metabolism and hypercholesterolemia.

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

Background and aimsEpidemiological 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.MethodsGSTM1 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.ResultsGSTM1 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.ConclusionsChronic 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.Graphical

Melatonin as a Repairing Agent in Cadmium- and Free Fatty Acid-Induced Lipotoxicity.

(1) Background: Cadmium (Cd) is a potentially toxic element with a long half-life in the human body (20-40 years). Cytotoxicity mechanisms of Cd include increased levels of oxidative stress and apoptotic signaling, and recent studies have suggested that these aspects of Cd toxicity contribute a role in the pathobiology of non-alcoholic fatty liver disease (NAFLD), a highly prevalent ailment associated with hepatic lipotoxicity and an increased generation of reactive oxygen species (ROS). In this study, Cd toxicity and its interplay with fatty acid (FA)-induced lipotoxicity have been studied in intestinal epithelium and liver cells; the cytoprotective function of melatonin (MLT) has been also evaluated. (2) Methods: human liver cells (HepaRG), primary murine hepatocytes and Caco-2 intestinal epithelial cells were exposed to CdCl2 before and after induction of lipotoxicity with oleic acid (OA) and/or palmitic acid (PA), and in some experiments, FA was combined with MLT (50 nM) treatment. (3) Results: CdCl2 toxicity was associated with ROS induction and reduced cell viability in both the hepatic and intestinal cells. Cd and FA synergized to induce lipid droplet formation and ROS production; the latter was higher for PA compared to OA in liver cells, resulting in a higher reduction in cell viability, especially in HepaRG and primary hepatocytes, whereas CACO-2 cells showed higher resistance to Cd/PA-induced lipotoxicity compared to liver cells. MLT showed significant protection against Cd toxicity either considered alone or combined with FFA-induced lipotoxicity in primary liver cells. (4) Conclusions: Cd and PA combine their pro-oxidant activity to induce lipotoxicity in cellular populations of the gut-liver axis. MLT can be used to lessen the synergistic effect of Cd-PA on cellular ROS formation.

AML Inhibits Lipid Uptake By the Liver through an HGF/c-MET Dependent Mechanism Which Results in an Increased Supply of Free Fatty Acid to the Tumor

Acute myeloid leukemia (AML) requires a high metabolic turnover to allow for constant expansion. We and others have previously shown that primary human AML blasts rely on fatty acid (FA)-oxidation for survival and proliferation. (Shafat MS, et al. 2017, Jones CL, et al. 2018). The liver plays a major role in carbohydrate, protein, amino acid, and lipid metabolism (Hodson L, and Gunn PJ. 2019). Given that AML is a FA dependent tumor, we have studied the interactions between AML and the liver, and specifically how these affect lipid metabolism, the availability of pro-tumoral FFA and ultimately the mechanism by which this supports AML growth. To understand the role of lipid metabolism we used two syngeneic models of AML (HOXA9/Meis1 or MN1). When engrafted with AML cells, mice body weight during the latter stages of disease. After sacrifice, the size of epididymal and inguinal fat pads were significantly reduced in mice with AML compared to controls. Mice with AML also had elevated free fatty acid (FFA) and reduced glucose in the plasma. We and others have shown that AML induces lipolysis of white adipose tissue for uptake by blasts and resulting in tumor expansion (Shafat MS, et al. 2017, Ye H, et al. 2016). Since the liver is the master regulator of available FA in the plasma, we isolated livers from AML engrafted mice. Transcriptomics revealed down-regulation of genes involved in FA uptake and metabolism. RT-qPCR of RNA isolated from liver samples confirmed these results showing down-regulation of FA transport proteins, (FABP1, SLC27A2, SLC27A5, and CD36) and FA metabolism genes (CPT1, ACADM, and HMGCS2). Furthermore, primary hepatocytes isolated and cultured with AML conditioned media showed a down-regulation of FA uptake and metabolism genes. These data support the hypothesis that AML secretes a factor that alters lipid metabolism in the liver. To discover the factor that AML secretes, and which regulates FA uptake and metabolism in the liver, a proteome profile of the serum from AML engrafted mice was compared to conditioned media from AML cultured cells. Pathway analysis revealed hepatocyte growth factor (HGF), IL-1B and Granulocyte colony stimulating factor (G-CSF) as possible key factors involved in liver FA metabolism (Jing Y, et al. 2019, Kaibori M, et al. 2002). In vitro gene expressions analysis of primary hepatocytes revealed that addition of HGF to the medium, but not Il-1b or G-CSF, inhibited expression of FA uptake genes. In addition, using Seahorse mitochondrial stress kit we confirmed that primary hepatocytes decreased FA oxidation when cultured with AML conditioned media or HGF. Next, we confirmed that AML derived HGF was responsible for inhibition of hepatocyte FA genes by using a neutralizing antibody to HGF which blocked the transcriptional changes previously observed in hepatocytes cultured in AML conditioned media. This showed that AML derived HGF downregulates hepatocyte FA uptake and metabolism. Finally, to determine the role of AML in the uptake of FA by hepatocytes, cultures of primary murine hepatocytes were pretreated with AML conditioned media or HGF with and without neutralizing antibody. A BODIPY conjugated long chain FA was then added to the culture. Images analysis showed hepatocytes had reduced lipid uptake in the presence of AML condition media or HGF, but this was restored with the addition of HGF neutralizing antibody. Here we report that AML inhibition of lipid uptake in the liver, through an HGF dependent mechanism, increases the availability of plasma FA and forms a fundamental part of the AML disease. Ongoing work to develop our understanding of how AML increases access to FA to aid proliferation, may aid in the development of new therapeutics to target liver metabolism in combination with other AML therapies.

Stimuli-Responsive Pressure-Strain Sensor-Based Conductive Hydrogel for Alleviated Non-alcoholic Fatty Liver Disease by Scavenging Reactive Oxygen Species in Adipose Tissue.

A visible light- and reactive oxygen species (ROS)-responsive pressure/strain sensor based on carbon dot (CD)-loaded conductive hydrogel was developed for detecting high-fat diet (HFD) and preventing the risk of non-alcoholic fatty liver disease. The designed nanoparticle consisted of a diselenide polymer dot (dsPD) loaded with a visible light-responsive CD to form dsPD@CD (DSCD). The influence of visible light irradiation and ROS on DSCD facilitated the electron transport, enhancing the conductivity of DSCD-embedded hydrogel (DSCD hydrogel) from 1.3 to 35.9 mS/m. Alternatively, the tensile modulus of the DSCD hydrogel enhanced to 223% after light-induced ROS treatment, which simultaneously impacted the capacitive response (120%). The hydrogel implantation into inguinal white adipose tissue of HFD mice showed 82% higher conductivity and 83% enhanced pressure sensing response to HFD-generated high ROS levels compared with the normal diet-fed mice. Additionally, the ROS scavenging activity of DSCD hydrogel was confirmed by the downregulation of ROS-responsive genes, such as Sod2, Nrf2, and catalase (Cat) in murine primary hepatocytes isolated from fatty liver-induced mice. In addition, in vivo animal studies also confirmed the suppression of hepatic lipogenesis, as shown by decreased Pparγ and Fasn expression and hypertrophy of adipocytes in HFD mice. The distinguishable real-time wireless resistance response observed with pressure sensing indicates the potential application of the device for monitoring the risk of non-alcoholic fatty liver disease. STATEMENT OF SIGNIFICANCE: : A visible-light-induced ROS-responsive carbon dot-loaded conductive hydrogel was developed for the detection of HFD-induced alterations in ROS levels by evaluating the conductivity and electrochemical responses with applied pressure/strain. The implanted hydrogel facilitates the recovery of the inflated adipocytes induced by NAFLD, which reduces fat accumulation in the liver, preventing the risk of NAFLD. Real-time detection based on the resistance response during local compression of the hydrogel is possibly performed utilizing a wireless sensing device, demonstrating the ease of NAFLD monitoring.

Wnt/β-catenin and NFκB signaling synergize to trigger growth factor-free regeneration of adult primary human hepatocytes.

The liver has a remarkable capacity to regenerate, which is sustained by the ability of hepatocytes to act as facultative stem cells that, while normally quiescent, re-enter the cell cycle after injury. Growth factor signaling is indispensable in rodents, whereas Wnt/β-catenin is not required for effective tissue repair. However, the molecular networks that control human liver regeneration remain unclear. Organotypic 3D spheroid cultures of primary human or murine hepatocytes were used to identify the signaling network underlying cell cycle re-entry. Furthermore, we performed chemogenomic screening of a library enriched for epigenetic regulators and modulators of immune function to determine the importance of epigenomic control for human hepatocyte regeneration. Our results showed that, unlike in rodents, activation of Wnt/β-catenin signaling is the major mitogenic cue for adult primary human hepatocytes. Furthermore, we identified TGFβ inhibition and inflammatory signaling through NF-κB as essential steps for the quiescent-to-regenerative switch that allows Wnt/β-catenin-induced proliferation of human cells. In contrast, growth factors, but not Wnt/β-catenin signaling, triggered hyperplasia in murine hepatocytes. High-throughput screening in a human model confirmed the relevance of NFκB and revealed the critical roles of polycomb repressive complex 2, as well as of the bromodomain families I, II, and IV. This study revealed a network of NFκB, TGFβ, and Wnt/β-catenin that controls human hepatocyte regeneration in the absence of exogenous growth factors, identified novel regulators of hepatocyte proliferation, and highlighted the potential of organotypic culture systems for chemogenomic interrogation of complex physiological processes.

Bempedoic Acid Unveils Therapeutic Potential in Non-Alcoholic Fatty Liver Disease: Suppression of the Hepatic PXR-SLC13A5/ACLY Signaling Axis.

The hepatic SLC13A5/SLC25A1-ATP-dependent citrate lyase (ACLY) signaling pathway, responsible for maintaining the citrate homeostasis, plays a crucial role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Bempedoic acid (BA), an ACLY inhibitor commonly used for managing hypercholesterolemia, has shown promising results in addressing hepatic steatosis. This study aimed to elucidate the intricate relationships in processes of hepatic lipogenesis among SLC13A5, SLC25A1, and ACLY and to examine the therapeutic potential of BA in NAFLD, providing insights into its underlying mechanism. In murine primary hepatocytes and HepG2 cells, the silencing or pharmacological inhibition of SLC25A1/ACLY resulted in significant upregulation of SLC13A5 transcription and activity. This increase in SLC13A5 activity subsequently led to enhanced lipogenesis, indicating a compensatory role of SLC13A5 when the SLC25A1/ACLY pathway was inhibited. However, BA effectively counteracted this upregulation, reduced lipid accumulation, and ameliorated various biomarkers of NAFLD. The disease-modifying effects of BA were further confirmed in NAFLD mice. Mechanistic investigations revealed that BA could reverse the elevated transcription levels of SLC13A5 and ACLY, and the subsequent lipogenesis induced by PXR activation in vitro and in vivo. Importantly, this effect was diminished when PXR was knocked down, suggesting the involvement of the hepatic PXR-SLC13A5/ACLY signaling axis in the mechanism of BA action. In conclusion, SLC13A5-mediated extracellular citrate influx emerges as an alternative pathway to SLC25A1/ACLY in the regulation of lipogenesis in hepatocytes, BA exhibits therapeutic potential in NAFLD by suppressing the hepatic PXR-SLC13A5/ACLY signaling axis, while PXR, a key regulator in drug metabolism may be involved in the pathogenesis of NAFLD. SIGNIFICANCE STATEMENT: This work describes that bempedoic acid, an ATP-dependent citrate lyase (ACLY) inhibitor, ameliorates hepatic lipid accumulation and various hallmarks of non-alcoholic fatty liver disease. Suppression of hepatic SLC25A1-ACLY pathway upregulates SLC13A5 transcription, which in turn activates extracellular citrate influx and the subsequent DNL. Whereas in hepatocytes or the liver tissue challenged with high energy intake, bempedoic acid reverses compensatory activation of SLC13A5 via modulating the hepatic PXR-SLC13A5/ACLY axis, thereby simultaneously downregulating SLC13A5 and ACLY.

Protective effects of the succinate/SUCNR1 axis on damaged hepatocytes in NAFLD

ObjectiveSuccinate and succinate receptor 1 (SUCNR1) are linked to fibrotic remodeling in models of non-alcoholic fatty liver disease (NAFLD), but whether they have roles beyond the activation of hepatic stellate cells remains unexplored. We investigated the succinate/SUCNR1 axis in the context of NAFLD specifically in hepatocytes. MethodsWe studied the phenotype of wild-type and Sucnr1−/− mice fed a choline-deficient high-fat diet to induce non-alcoholic steatohepatitis (NASH), and explored the function of SUCNR1 in murine primary hepatocytes and human HepG2 cells treated with palmitic acid. Lastly, plasma succinate and hepatic SUCNR1 expression were analyzed in four independent cohorts of patients in different NAFLD stages. ResultsSucnr1 was upregulated in murine liver and primary hepatocytes in response to diet-induced NASH. Sucnr1 deficiency provoked both beneficial (reduced fibrosis and endoplasmic reticulum stress) and detrimental (exacerbated steatosis and inflammation and reduced glycogen content) effects in the liver, and disrupted glucose homeostasis. Studies in vitro revealed that hepatocyte injury increased Sucnr1 expression, which when activated improved lipid and glycogen homeostasis in damaged hepatocytes. In humans, SUCNR1 expression was a good determinant of NAFLD progression to advanced stages. In a population at risk of NAFLD, circulating succinate was elevated in patients with a fatty liver index (FLI) ≥60. Indeed, succinate had good predictive value for steatosis diagnosed by FLI, and improved the prediction of moderate/severe steatosis through biopsy when added to an FLI algorithm. ConclusionsWe identify hepatocytes as target cells of extracellular succinate during NAFLD progression and uncover a hitherto unknown function for SUCNR1 as a regulator of hepatocyte glucose and lipid metabolism. Our clinical data highlight the potential of succinate and hepatic SUCNR1 expression as markers to diagnose fatty liver and NASH, respectively.

Boosting intracellular sodium selectively kills hepatocarcinoma cells and induces hepatocellular carcinoma tumor shrinkage in mice

Pharmacological treatments for advanced hepatocellular carcinoma (HCC) have a partial efficacy. Augmented Na+ content and water retention are observed in human cancers and offer unexplored targets for anticancer therapies. Na+ levels are evaluated upon treatments with the antibiotic cation ionophore Monensin by fluorimetry, ICP-MS, 23Na-MRI, NMR relaxometry, confocal or time-lapse analysis related to energy production, water fluxes and cell death, employing both murine and human HCC cell lines, primary murine hepatocytes, or HCC allografts in NSG mice. Na+ levels of HCC cells and tissue are 8-10 times higher than that of healthy hepatocytes and livers. Monensin further increases Na+ levels in HCC cells and in HCC allografts but not in primary hepatocytes and in normal hepatic and extrahepatic tissue. The Na+ increase is associated with energy depletion, mitochondrial Na+ load and inhibition of O2 consumption. The Na+ increase causes an enhancement of the intracellular water lifetime and death of HCC cells, and a regression and necrosis of allograft tumors, without affecting the proliferating activity of either HCCs or healthy tissues. These observations indicate that HCC cells are, unlike healthy cells, energetically incapable of compensating and surviving a pharmacologically induced Na+ load, highlighting Na+ homeostasis as druggable target for HCC therapy.

FKBP12 inhibits hepcidin expression by modulating BMP receptors interaction and ligand responsiveness in hepatocytes.

The expression of the iron regulatory hormone hepcidin in hepatocytes is regulated by the BMP-SMAD pathway through the type I receptors ALK2 and ALK3, the type II receptors ACVR2A and BMPR2, and the ligands BMP2 and BMP6. We previously identified the immunophilin FKBP12 as a new hepcidin inhibitor that acts by blocking ALK2. Both the physiologic ALK2 ligand BMP6 and the immunosuppressive drug Tacrolimus (TAC) displace FKBP12 from ALK2 and activate the signaling. However, the molecular mechanism whereby FKBP12 regulates BMP-SMAD pathway activity and thus hepcidin expression remains unclear. Here, we show that FKBP12 acts by modulating BMP receptor interactions and ligand responsiveness. We first demonstrate that in primary murine hepatocytes TAC regulates hepcidin expression exclusively via FKBP12. Downregulation of the BMP receptors reveals that ALK2, to a lesser extent ALK3, and ACVR2A are required for hepcidin upregulation in response to both BMP6 and TAC. Mechanistically, TAC and BMP6 increase ALK2 homo-oligomerization and ALK2-ALK3 hetero-oligomerization and the interaction between ALK2 and the type II receptors. By acting on the same receptors, TAC and BMP6 cooperate in BMP pathway activation and hepcidin expression both in vitro and in vivo. Interestingly, the activation state of ALK3 modulates its interaction with FKBP12, which may explain the cell-specific activity of FKBP12. Overall, our results identify the mechanism whereby FKBP12 regulates the BMP-SMAD pathway and hepcidin expression in hepatocytes, and suggest that FKBP12-ALK2 interaction is a potential pharmacologic target in disorders caused by defective BMP-SMAD signaling and characterized by low hepcidin and high BMP6 expression.

STING mediates hepatocyte pyroptosis in liver fibrosis by Epigenetically activating the NLRP3 inflammasome

The activation of stimulator of interferon genes (STING) and NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis signaling pathways represent two distinct central mechanisms in liver disease. However, the interconnections between these two pathways and the epigenetic regulation of the STING-NLRP3 axis in hepatocyte pyroptosis during liver fibrosis remain unknown. STING and NLRP3 inflammasome signaling pathways are activated in fibrotic livers but are suppressed by Sting knockout. Sting knockout ameliorated hepatic pyroptosis, inflammation, and fibrosis. In vitro, STING induces pyroptosis in primary murine hepatocytes by activating the NLRP3 inflammasome. H3K4-specific histone methyltransferase WD repeat-containing protein 5 (WDR5) and DOT1-like histone H3K79 methyltransferase (DOT1L) are identified to regulate NLRP3 expression in STING-overexpressing AML12 hepatocytes. WDR5/DOT1L-mediated histone methylation enhances interferon regulatory transcription factor 3 (IRF3) binding to the Nlrp3 promoter and promotes STING-induced Nlrp3 transcription in hepatocytes. Moreover, hepatocyte-specific Nlrp3 deletion and downstream Gasdermin D (Gsdmd) knockout attenuate hepatic pyroptosis, inflammation, and fibrosis. RNA-sequencing and metabolomics analysis in murine livers and primary hepatocytes show that oxidative stress and metabolic reprogramming might participate in NLRP3-mediated hepatocyte pyroptosis and liver fibrosis. The STING-NLRP3-GSDMD axis inhibition suppresses hepatic ROS generation. In conclusion, this study describes a novel epigenetic mechanism by which the STING-WDR5/DOT1L/IRF3-NLRP3 signaling pathway enhances hepatocyte pyroptosis and hepatic inflammation in liver fibrosis.

Mitophagy and endoplasmic reticulum-phagy accelerated by a p62 ZZ ligand alleviates paracetamol-induced hepatotoxicity.

Paracetamol (acetaminophen)-induced hepatotoxicity is the leading cause of drug-induced liver injury worldwide. Autophagy is a degradative process by which various cargoes are collected by the autophagic receptors such as p62/SQSTM1/Sequestosome-1 for lysosomal degradation. Here, we investigated the protective role of p62-dependent autophagy in paracetamol-induced liver injury. Paracetamol-induced hepatotoxicity was induced by a single i.p. injection of paracetamol (500 mg·kg-1 ) in C57/BL6 male mice. YTK-2205 (20 mg·kg-1 ), a p62 agonist targeting ZZ domain, was co- or post-administered with paracetamol. Western blotting and immunocytochemistry were performed to explore the mechanism. N-terminal arginylation of the molecular chaperone calreticulin retro-translocated from the endoplasmic reticulum (ER) was induced in the livers undergoing paracetamol-induced hepatotoxicity, and YTK-2205 exhibited notable therapeutic efficacy in acute hepatotoxicity as assessed by the levels of serum alanine aminotransferase and hepatic necrosis. This efficacy was significantly attributed to accelerated degradation of ubiquitin (Ub) conjugates as well as damaged mitochondria (mitophagy) and endoplasmic reticulum (ER-phagy). In primary murine hepatocytes treated with paracetamol, YTK-2205 induced the co-localization of p62+ LC3+ phagophores to the sites of mitophagy and ER-phagy. A similar activity of YTK-2205 was observed with N-acetyl-p-benzoquinone imine, a putative toxic metabolite of paracetamol in Hep3B cells. Our results elucidated that p62-dependent autophagy plays a key role in the removal of cytotoxic materials such as damaged mitochondria in paracetamol-induced hepatotoxicity. Small molecule ligands to p62 may be developed into drugs to treat this pathological condition.

Murine Fibroblasts and Primary Hepatocytes as Tools When Studying the Efficacy of Potential Therapies for Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I) is a metabolic genetic disease caused by the deficiency of a lysosomal enzyme involved in glycosaminoglycans (GAGs) degradation. MPS I cells have a constant level of GAG synthesis, but disturbed degradation means that GAGs accumulate progressively, impairing cell metabolism. GAG metabolism can be modulated by flavonoids, and these are being studied as therapeutics for MPS. We have optimised the protocol for obtaining fibroblasts and hepatocytes from the MPS I murine model and characterised the cells for their suitability as an in vitro model for testing compounds with therapeutic potential. Methods: Murine primary hepatocytes and fibroblasts were used as a cellular model to study the effect of genistein, biochanin A, and kaempferol on the modulation of the GAG synthesis process. Flavonoids were used individually as well as in two-component mixtures. There were no statistically significant differences in GAG synthesis levels from cell types obtained from either wild-type or MPS I mice. We also showed that MPS I fibroblasts and hepatocytes store GAGs, which makes them useful in vitro models for testing the effectiveness of substrate reduction therapies. Furthermore, tested flavonoids had a different impact on GAG synthesis depending on cell type and whether they were used alone or in a mixture. The tested flavonoids reduce GAG synthesis more effectively in fibroblasts than in hepatocytes, regardless of whether they are used individually or in a mixture. Flavonoids modulate the level of GAG synthesis differently depending on cell types, therefore in vitro experiments performed to assess the effectiveness of potential therapies for metabolic diseases should be carried out using more than one cell model, and only such an approach will allow for full answering scientific questions.

Cell-Specific Transport and Thyroid Hormone Receptor Isoform Selectivity Account for Hepatocyte-Targeted Thyromimetic Action of MGL-3196.

Thyroid hormones (THs) and TH receptor-beta (TRβ) reduce hepatic triglycerides, indicating a therapeutic potential for TH analogs in liver steatosis. To avoid adverse extrahepatic, especially TRα-mediated effects such as tachycardia and bone loss, TH analogs with combined TRβ and hepatocyte specificity are desired. MGL-3196 is a new TH analog that supposedly meets these criteria. Here, we characterize the thyromimetic potential of MGL-3196 in cell-based assays and address its cellular uptake requirements. We studied the contribution of liver-specific organic anion transporters (OATP)1B1 and 1B3 to MGL-3196 action. The TR isoform-specific efficacy of MGL-3196 compared with 3,5,3'-triiodothyronine (T3) was determined with luciferase assays and gene expression analysis in OATP1B1 and OATP1B3 and TRα- or TRβ-expressing cells and in primary murine hepatocytes (PMHs) from wild-type and TRβ knockout mice. We measured the oxygen consumption rate to compare the effects of MGL-3196 and T3 on mitochondrial respiration. We identified OATP1B1 as the primary transporter for MGL-3196. MGL-3196 had a high efficacy (90% that of T3) in activating TRβ, while the activation of TRα was only 25%. The treatment of PMHs with T3 and MGL-3196 at EC50 resulted in a similar induction of Dio1 and repression of Serpina7. In HEK293 cells stably expressing OATP1B1, MGL-3196 had comparable effects on mitochondrial respiration as T3. These data indicate that MGL-3196's hepatic thyromimetic action, the basis for its therapeutic use, results from a combination of hepatocyte-specific transport by OATP1B1 and the selective activation of TRβ over TRα.

Core-Shell Structured Chitosan-Polyethylenimine Nanoparticles for Gene Delivery: Improved Stability, Cellular Uptake, and Transfection Efficiency.

The delivery of nucleic acids relies on vectors that condense and encapsulate their cargo. Especially nonviral gene delivery systems are of increasing interest. However, low transgene expression levels and limited tolerability of these systems remain a challenge. The improvement of nucleic acid delivery using depolymerized chitosan-polyethylenimine DNA complexes (dCS-PEI/DNA) is investigated. The secore complexes are further combined with chitosan-based shells and functionalized with polyethylene glycol (PEG) and cell penetrating peptides. This modular approach allows to evaluate the effect of functional shell components on physicochemical particle characteristics and biological effects. The optimized ternary complex combines a core-dCS-linear PEI/DNA complex with a shell consisting of dCS-PEG-COOH, which results in improved nucleic acid encapsulation, cellular uptake and transfection potency in human hepatoma HuH-7cells and murine primary hepatocytes. Effects on transgene expression are confirmed in wild-type mice following retrograde intrabiliary infusion. After administration of only 100 ng complexed DNA, ternary complexes induced a high reporter gene signal for three days. It is concluded that ternary coreshell structured nanoparticles comprising functionalized chitosan can be used for in vitro andin vivo gene delivery.

Adeno-associated virus serotype 2 capsid variants for improved liver-directed gene therapy.

Current liver-directed gene therapies look for adeno-associated virus (AAV) vectors with improved efficacy. With this background, capsid engineering is explored. Whereas shuffled capsid library screenings have resulted in potent liver targeting variants with one first vector in human clinical trials, modifying natural serotypes by peptide insertion has so far been less successful. Here, we now report on two capsid variants, MLIV.K and MLIV.A, both derived from a high-throughput in vivo AAV peptide display selection screen in mice. The variants transduce primary murine and human hepatocytes at comparable efficiencies, a valuable feature in clinical development, and show significantly improved liver transduction efficacy, thereby allowing a dose reduction, and outperform parental AAV2 and AAV8 in targeting human hepatocytes in humanized mice. The natural heparan sulfate proteoglycan binding ability is markedly reduced, a feature that correlates with improved hepatocyte transduction. A further property that might contribute to the improved transduction efficacy is the lower capsid melting temperature. Peptide insertion also caused a moderate change in sensitivity to human sera containing anti-AAV2 neutralizing antibodies, revealing the impact of epitopes located at the basis of the AAV capsid protrusions. In conclusion, MLIV.K and MLIV.A are AAV peptide display variants selected in immunocompetent mice with improved hepatocyte tropism and transduction efficiency. Because these features are maintained across species, MLIV variants provide remarkable potential for translation of therapeutic approaches from mice to men.