Primary Cultured Mouse Hepatocytes Research Articles

Apolipoprotein B Secretion Assay from Primary Hepatocytes.

Apolipoprotein B (APOB) is the primary structural protein of atherogenic lipoproteins, which drive atherogenesis and thereby lead to deadly cardiovascular diseases (CVDs). Plasma levels of APOB-containing lipoproteins are tightly modulated by LDL receptor-mediated endocytic trafficking and cargo receptor-initiated exocytic route; the latter is much less well understood. This protocol aims to present an uncomplicated yet effective method for detecting APOB/lipoprotein secretion. We perform primary mouse hepatocyte isolation and culture coupled with well-established techniques such as immunoblotting for highly sensitive, specific, and semi-quantitative analysis of the lipoprotein secretion process. Its inherent simplicity facilitates ease of operation, rendering it a valuable tool widely utilized to explore the intricate landscape of cellular lipid metabolism and unravel the mechanistic complexities underlying lipoprotein-related diseases. Key features • A pipeline for the isolation and subsequent culture of mouse primary hepatocytes. • A procedure for tracking the secretion of APOB-containing lipoproteins. • A rapid and sensitive assay for detecting the APOB level based on immunoblotting.

Systematic screening of hepatoprotective components from traditional Chinese medicine: Zuojin Pill as an example

Ethnopharmacological relevanceZuojin Pill (ZJP), composed of Coptis chinensis Franch. and Euodia ruticarpa (A. Juss.) Benth. in a mass ratio of 6:1, is a famous traditional Chinese medicine (TCM) formula recorded in “Danxi's Experiential Therapy”, an ancient medical book from the Ming Dynasty of China. It is used to treat liver fire invading the stomach, which is caused by liver stagnation transforming into fire and disharmony between the liver and stomach. Aim of the studyTo develop a systematic strategy to screen hepatoprotective components from TCM using ZJP as a model sample. Materials and methodsA CCl4-induced mouse model of acute liver injury was used for the verification of the hepatoprotective effects of ZJP. UPLC-Q-Exactive Plus Orbitrap MS/MS was used for the identification of the components in mouse serum after intragastric administration of ZJP. The hepatoprotective activities of the components found in mouse serum were tested in primary cultured mouse hepatocytes induced by CCl4. ResultsNine components with significant hepatoprotective activity including berberine, epiberberine, coptisine, palmatine, jatrorrhizine, rutaecarpin, dehydroevodiamine, evocarpine and chlorogenic acid were successfully screened out. ConclusionsOur developed strategy has the advantages of high efficiency and low cost, and would provide a powerful tool for screening potential hepatoprotective components from TCM.

LRG1 inhibits hepatic macrophage activation by enhancing TGF-β1 signaling to alleviate MAFLD in mice

To explore the effect of leucine-rich α-2-glycoprotein (LRG1) derived from hepatocytes on activation of hepatic M1 Kupffer cells. A metabolic dysfunction-associated fatty liver disease (MAFLD) model was established in BALB/c mice by high-fat diet (HFD) feeding for 16 weeks. Oleic acid was used to induce steatosis in primary cultures of mouse hepatocytes. The mRNA and protein expressions of LRG1 in mouse liver tissues and hepatocytes were detected by real-time PCR and Western blotting. Primary hepatic macrophages were stimulated with the conditioned medium (CM) from steatotic hepatocyte along with LRG1 or transforming growth factor-β1 (TGF-β1), or both for 24 h, and the expression levels of inducible nitric oxide synthase (iNOS) was detected with Western botting, and the mRNA expressions of iNOS, chemokine ligand 1 (CXCL-1) and interleukin-1β (IL-1β) were measured by RT-PCR. The MAFLD mice were injected with LRG1 (n=6), TGF-β1 (n=6), or both (n=6) through the caudal vein, and the live tissues were collected for HE staining and immumohistochemical detection of F4/80 expression; the mRNA expressions of iNOS, CXCL-1 and IL-1β in liver tissues were detected using RT-PCR. The mRNA and protein expression levels of LRG1 were significantly downregulated in the liver tissues of MAFLD mice and steatotic hepatocytes (P < 0.05). Treatment of the hepatic macrophages with CM from steatosis hepatocytes significantly enhanced the mRNA expression levels of iNOS, CXCL-1 and IL-1β, and these changes were significantly inhibited by the combined treatment with TGF-β1 and LRG1 (P < 0.05). In MAFLD mice, injections with either LRG1 or TGF-β1 alone reduced hepatic lipid deposition and intrahepatic macrophage infiltration, and these effects were significantly enhanced by their combined treatment, which also more strongly inhibited the mRNA expression levels of iNOS, CXCL-1 and IL-1β (P < 0.05). LRG1 inhibits hepatic macrophage infiltration by enhancing TGF-β1 signaling to alleviate fatty liver inflammation in MAFLD mice.

Methods for Monitoring Mitochondrial Biogenesis and Turnover in Cultured Hepatocytes and Mouse Liver Using MitoTimer Reporter Assay.

Mitochondrial biogenesis and turnover rate are critical to maintain homeostasis of the intracellular mitochondrial pool. Altered mitochondrial biogenesis and mitophagy are closely related to many chronic diseases, highlighting the importance of mitochondrial stasis in various pathological conditions including liver diseases. We describe a detailed protocol for monitoring mitochondrial lifecycle in primary cultured mouse hepatocytes and mouse liver using the dual color fluorescence-based imaging of MitoTimer. Three types of mitochondria were visualized in mouse hepatocytes: green-only mitochondria (newly synthesized mitochondria), red-only mitochondria (old/aging mitochondria), as well as the majority of yellow mitochondria (representing an intermediate stage of mitochondria). The ratio of red/green fluorescence in each cell will be used to track mitochondrial aging. Super-resolution microscopy analysis revealed that majority of mitochondria were spatially heterogeneous with proteins from simultaneous new synthesis, maturation, and turnover in hepatocytes. MitoTimer reporter assay can specifically target to mitochondria and be used to monitor mitochondrial biogenesis and maturation as well as turnover in vitro and in vivo.

MRG15 aggravates non-alcoholic steatohepatitis progression by regulating the mitochondrial proteolytic degradation of TUFM.

How hepatic steatosis progresses to non-alcoholic steatohepatitis (NASH) is complicated and remains unclear. The mortality factor 4-like protein 1 (MORF4L1, also called MRG15) was previously identified as a master nuclear chromatin remodeler in the rhythmic regulation of lipid synthesis gene expression in the liver. Whether it also contributes to the progression from liver steatosis to NASH is unclear. We adopted 2 different murine NASH models, liver biopsies from patients with NASH, and primary mouse and human hepatocyte cultures for functional examination of MRG15 in NASH progression. Immunoprecipitation-mass spectrometry was applied to identify protein partners of MRG15, and CRISPR targeting was used for gene depletion in liver cells invivo. The MRG15 level is increased in the livers of humans and mice with NASH. The inflammatory cytokines in NASH livers stabilize MRG15 by increasing its acetylation. Considerable amounts of MRG15 associate with the outer mitochondrial membrane, where it interacts with and deacetylates the mitochondrial Tu translation elongation factor (TUFM). Deacetylated TUFM, especially at the K82 and K91 sites, is subjected to accelerated degradation by the mitochondrial ClpXP protease system. Reduced liver TUFM consequently results in impaired mitophagy, increased oxidative stress and activation of the NLRP3 inflammasome pathway. Blocking MRG15 expression protects the liver from NASH progression by increasing the stability of liver TUFM. Liver samples from patients with NASH also display a clear reduction in TUFM level, which correlates with increased MRG15 expression. Collectively, these findings uncover a mitochondrial MRG15-TUFM regulatory pathway that contributes significantly to progression from simple steatosis to NASH, and which could potentially be targeted to treat NASH. The incidence of non-alcoholic fatty liver diseaseand its progressive form non-alcoholic steatohepatitis (NASH) is increasing, posing a significant global health challenge. Herein,we have uncovered the importance of the MRG15-TUFM pathway in NASH development. This pathway is active in the mitochondria (energy powerhouse of the cell) and could be targeted for the treatment of NASH.

Arsenic-Induced Injury of Mouse Hepatocytes through Lysosome and Mitochondria: An In Vitro Study.

Background and Aims The cellular mechanism of liver injury related to arsenic toxicity is ill defined. It is thought that oxidative stress and mitochondrial dysfunction may play some role in arsenic-induced liver damage. In this study, we evaluated subcellular events within the primary cultured mouse hepatocytes when exposed to inorganic arsenic. Methods Primary cultured mouse hepatocytes were treated with 10 μM arsenic for different time periods. Reactive oxygen species (ROS) formation, functional changes of the lysosome and mitochondria, and mode of hepatocytes death were studied by laser confocal microscopy, fluorescence spectroscopy, and flow cytometry. Expression of proapoptotic member of the BCL-2 family of genes BAX and antiapoptotic BCL-2 mRNA expression were studied by real-time PCR. Cytochrome c expression was studied by Western blotting. Results Fluorescence spectroscopy as well as flow cytometric analysis revealed that arsenic-induced formation of ROS was time dependent. Confocal microscopy showed initiation of ROS formation from periphery of the hepatocytes at 30 min of arsenic exposure that progressed to central part of the hepatocytes at 3 h of arsenic exposure. The ROS formation was found to be NADPH oxidase (NOX) dependent. This low level of intracellular ROS induced lysosomal membrane permeabilization (LMP) and subsequently released cathepsin B to the cytosol. The LMP further increased intracellular ROS which in turn triggered induction of mitochondrial permeability transition (MPT). Pretreatment of hepatocytes with LMP inhibitor bafilomycin A (BafA) significantly decreased, and LMP inducer chloroquine (ChQ) significantly increased the production of ROS suggesting that LMP preceded enhanced ROS generation in response to arsenic. MPT was accompanied with increase in BAX : BCL2 mRNA ratio resulting in upregulation of caspase 3 and increased hepatocyte apoptosis. Conclusion Although arsenic-related oxidative liver injury is well established, neither the site of origin of ROS nor the early sequence of events in arsenic toxicity due to ROS is known. We believe that our study provides evidences elucidating the early sequence of events that culminates in the death of the mouse hepatocytes during arsenic exposure.

Regulation of Cytochrome P450 2a5 by Artemisia capillaris and 6,7-Dimethylesculetin in Mouse Hepatocytes.

Jaundice is a potentially fatal condition resulting from elevated serum bilirubin levels. For centuries, herbal remedies containing Artemisia capillaris Thunb. including the compound 6,7-dimethylesculetin (DE) have been used in Asia to prevent and treat jaundice in neonates. DE activates an important regulator of bilirubin metabolism, the constitutive androstane receptor (CAR), and increases bilirubin clearance. In addition, murine cytochrome P450 2a5 (Cyp2a5) is known to be involved in the oxidative metabolism of bilirubin. Moreover, treatment of mice with phenobarbital, a known inducer of both CAR and Cyp2a5, increases expression of Cyp2a5 suggesting a potential relationship between CAR and Cyp2a5 expression. The aim of this study is to investigate the influence of Artemisia capillaris and DE on the expression and regulatory control of Cyp2a5 and the potential involvement of CAR. Treatment of mouse hepatocytes in primary culture with DE (50 μM) significant increased Cyp2a5 mRNA and protein levels. In mice, Artemisia capillaris and DE treatment also increased levels of hepatic Cyp2a5 protein. Luciferase reporter assays showed that CAR increases Cyp2a5 gene transcription through a CAR response element in the Cyp2a5 gene promoter. Moreover, DE caused nuclear translocation of CAR in primary mouse hepatocytes and increased Cyp2a5 transcription in the presence of CAR. These results identify a potential CAR-mediated mechanism by which DE regulates Cyp2a5 gene expression and suggests that DE may enhance bilirubin clearance by increasing Cyp2a5 levels. Understanding this process could provide an opportunity for the development of novel therapies for neonatal and other forms of jaundice.

Remifentanil preconditioning promotes liver regeneration via upregulation of β-arrestin 2/ERK/cyclin D1 pathway

Remifentanil is a potent, short-acting opioid analgesic drug that can protect tissues from ischemia and reperfusion injury though anti-inflammatory effects. However, the utility of remifentanil in liver regeneration after hepatectomy is not known. Using a 70% hepatectomy mouse model (PHx), we found that preconditioning animals with 4 μg/kg remifentanil enhanced liver regeneration through supporting hepatocyte proliferation but not through anti-inflammatory effects. These effects were also phenocopied in vitro where 40 mM remifentanil promoted the proliferation of primary mouse hepatocyte cultures. We further identified that remifentanil treatment increased the expression of β-arrestin 2 in vivo and in vitro. Demonstrating specificity, remifentanil preconditioning failed to promote liver regeneration in liver-specific β-arrestin 2 knockout (CKO) mice subjected to PHx. While remifentanil increased the expression of activated (phosphorylated)-ERK and cyclin D1 in PHx livers, their levels were not significantly changed in remifentanil-treated CKO mice nor in WT mice pretreated with the ERK inhibitor U0126. Our findings suggest that remifentanil promotes liver regeneration via upregulation of a β-arrestin 2/ERK/cyclin D1 axis, with implications for improving regeneration process after hepatectomy.

Maresin 1 protects the liver against ischemia/reperfusion injury via the ALXR/Akt signaling pathway

BackgroundHepatic ischemia/reperfusion (I/R) injury can be a major complication following liver surgery contributing to post-operative liver dysfunction. Maresin 1 (MaR1), a pro-resolving lipid mediator, has been shown to suppress I/R injury. However, the mechanisms that account for the protective effects of MaR1 in I/R injury remain unknown.MethodsWT (C57BL/6J) mice were subjected to partial hepatic warm ischemia for 60mins followed by reperfusion. Mice were treated with MaR1 (5-20 ng/mouse), Boc2 (Lipoxin A4 receptor antagonist), LY294002 (Akt inhibitor) or corresponding controls just prior to liver I/R or at the beginning of reperfusion. Blood and liver samples were collected at 6 h post-reperfusion. Serum aminotransferase, histopathologic changes, inflammatory cytokines, and oxidative stress were analyzed to evaluate liver injury. Signaling pathways were also investigated in vitro using primary mouse hepatocyte (HC) cultures to identify underlying mechanisms for MaR1 in liver I/R injury.ResultsMaR1 treatment significantly reduced ALT and AST levels, diminished necrotic areas, suppressed inflammatory responses, attenuated oxidative stress and decreased hepatocyte apoptosis in liver after I/R. Akt signaling was significantly increased in the MaR1-treated liver I/R group compared with controls. The protective effect of MaR1 was abrogated by pretreatment with Boc2, which together with MaR1-induced Akt activation. MaR1-mediated liver protection was reversed by inhibition of Akt.ConclusionsMaR1 protects the liver against hepatic I/R injury via an ALXR/Akt signaling pathway. MaR1 may represent a novel therapeutic agent to mitigate the detrimental effects of I/R-induced liver injury.

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca2+, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD+] ratio, which was corroborated by using the NADH/NAD+ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD+ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD+ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.

HEPATOCYTE APOPTOSIS INDUCTION BY ACETAMINOPHEN THROUGH MODULATION OF CASPASE/BAX PATHWAY IN MICE

Objective: Acetaminophen (APAP) overdose contributes to liver damage through modulation of pro-apoptotic processing. This study evaluated the involvement of caspase/Bax factors in APAP hepatotoxicity in vivo and in vitro.&#x0D; Methods: The involvement of caspase/Bax factors in APAP hepatotoxicity was evaluated in BALB/c mice and on isolated primary mouse hepatocytes. In vitro MTT assay was carried out on primary cultured mouse hepatocytes treated with APAP (2.5, 5, 10 mmol) and Annexin V/PI staining was employed to cell suspension for imaging under fluorescence microscopy. In addition, caspase-3 concentrations were determined in cell lysates. In vivo, mice were treated with a toxic dose of APAP (700 mg/kg) and immunodetection of Bax was made by Western Blot. Vitamin C (Vit C) was used as a hepato-protectant due to its known antioxidant activities.&#x0D; Results: In vitro dose-dependent increase in mitochondrial electron transport capacity was evident in isolated mouse primary hepatocytes incubated with the high dose of APAP (10 mmol) compared to both nontreated cells and cells pre-treated with Vitamin C (Vit C) (0.5 mmol) (p&lt;0.05). Apoptosis was confirmed in hepatocytes through Annexin V staining after APAP treatment and the signal was reduced when hepatocytes were pre-treated with Vit C. In addition, caspase-3 concentration was decreased in cells pretreated with Vit C prior to APAP exposure. In vivo, Bax immunodetection utilizing western blotting was increased in mice treated with the toxic dose of APAP (700 mg/kg) and attenuated through pre-treatment with Vit C.&#x0D; Conclusion: Modulation of apoptotic caspase/Bax pathway is present in hepatocytes undergoing APAP-induced toxicity.

Untargeted Metabolomics Reveals Anaerobic Glycolysis as a Novel Target of the Hepatotoxic Antidepressant Nefazodone.

Mitochondrial damage is considered a hallmark of drug-induced liver injury (DILI). However, despite the common molecular etiology, the evolution of the injury is usually unpredictable, with some cases that are mild and reversible upon discontinuation of the treatment and others characterized by irreversible acute liver failure. This suggests that additional mechanisms of damage play a role in determining the progression of the initial insult. To uncover novel pathways potentially involved in DILI, we investigated in vitro the metabolic perturbations associated with nefazodone, an antidepressant associated with acute liver failure. Several pathways associated with ATP production, including gluconeogenesis, anaerobic glycolysis, and oxidative phosphorylation, were altered in human hepatocellular carcinoma-derived (Huh7) cells after 2-hour exposure to a 50 μM extracellular concentration of nefazodone. In the presence or absence of glucose, ATP production of Huh7 cells was glycolysis- and oxidative phosphorylation-dependent, respectively. In glucose-containing medium, nefazodone-induced ATP depletion from Huh7 cells was biphasic. Huh7 cells in glucose-free medium were more sensitive to nefazodone than those in glucose-containing medium, losing the biphasic inhibition. Nefazodone-induced ATP depletion in primary cultured mouse hepatocytes, mainly dependent on oxidative phosphorylation, was monophasic. At lower extracellular concentrations, nefazodone inhibited the oxygen consumption of Huh7 cells, whereas at higher extracellular concentrations, it also inhibited the extracellular acidification. ATP content was rescued by increasing the extracellular concentration of glucose. In conclusion, nefazodone has a dual inhibitory effect on mitochondrial-dependent and mitochondrial-independent ATP production. SIGNIFICANCE STATEMENT: Mitochondrial damage is a hallmark of drug-induced liver injury, yet other collateral alterations might contribute to the severity and evolution of the injury. Our in vitro study supports previous results arguing that a deficit in hepatic glucose metabolism, concomitant to the mitochondrial injury, might be cardinal in the prognosis of the initial insult to the liver. From a drug development standpoint, coupling anaerobic glycolysis and mitochondrial function assessment might increase the drug-induced liver injury preclinical screening performance.

Protein cross-linking in primary cultured mouse hepatocytes by dehydropyrrolizidine alkaloids: Structure–toxicity relationship

Pyrrolizidine alkaloids (PAs) are natural toxins found in about 3%–5% of flowering plants. Dehydropyrrolizidine alkaloids contain a double bond in 1, 2-position of the necine bases, including retronecine type PAs (RET-PAs) and their N-oxides (RET N-oxide-PAs), and otonecine type PAs (OTO-PAs), and are known for their significant hepatotoxicity. Most dehydropyrrolizidine alkaloids are metabolically activated by cytochrome P450 (CYP450) enzymes to generate active pyrroles, which further bind to proteins to form pyrrole–protein adducts (PPAs). Methods for predicting PA-induced liver injury are generally performed on in vitro models with extremely low activities of CYP450 enzymes, which is different from the situation in vivo. In this regard, primary cultured mouse hepatocytes, which showed comparable CYP450 activity with the in vivo models, were applied to illustrate the structure–toxicity relationship of 13 dehydropyrrolizidine alkaloids, namely, eight RET-PAs, three RET N-oxide-PAs, and two OTO-PAs. PA-induced cytotoxicity and PA-generated PPAs were analyzed in primary mouse hepatocytes treated with different PAs. Results showed that PA-induced toxicity was correlated with the amount of PA-generated PPAs. RET-PAs and OTO-PAs were generally more toxic than RET N-oxide-PAs and generated higher amount of PPAs. PPAs were utilized to evaluate the efficiency of metabolic activation and predict the toxic potencies of dehydropyrrolizidine alkaloids. The proposed model could be a new approach for toxicity evaluation and risk control of exposure to PAs.

Farnesoid X receptor (FXR) activation induces the antioxidant protein metallothionein 1 expression in mouse liver

Farnesoid X receptor (FXR) is a metabolic nuclear receptor, which protects liver from many endogenous and exogenous injuries. Metallothioneins (MTs) belong to a low-molecular-weight protein family involved in metal homeostasis and the regulation of hepatic oxidative stress. In the present study, we aimed to investigate the effect of FXR on hepatic MT1 expression and the underlying mechanism. C57BL/6 mice or primary cultured mouse hepatocytes were treated with the synthetic FXR ligand GW4064 or natural ligand CDCA. RNA-Sequencing (RNA-seq) analysis was performed to identify gene expression profile in the livers of mice treated with GW4064. Real-time PCR and Western blot were applied to determine the expression of MT1 and other FXR target genes in the livers of mice and primary hepatocytes treated with GW4064 and CDCA. Cellular and subcellular locations of MT1 in the livers of mice treated with GW4064 were examined using immunohistochemistry assay. FXR small interfering RNAs (siRNA) was transfected to silence FXR. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were utilized to confirm the regulation of MT1 gene promoter activity by FXR. RNA-seq analysis revealed that GW4064 treatment significantly induced MT1 expression in mouse liver. Consistently, MT1 expression in the hepatocytes of mouse livers and cultured hepatocytes was upregulated by GW4064 as well as CDCA. In addition, adenovirus-mediated overexpression of FXR markedly increased, while siRNA-mediated FXR silencing significantly suppressed MT1 expression in cultured hepatocytes. Luciferase reporter and ChIP assays further confirmed that the MT1 gene was under the direct control of FXR. Collectively, our findings demonstrate that MT1 is a novel target gene of FXR and may contribute to antioxidative capacity of FXR in liver diseases.

Role of Exosomes in Hepcidin Regulation in β-Thalassemia

β-thalassemia is characterized by ineffective erythropoiesis and iron overload. Ineffective erythropoiesis causes iron overload by suppressing hepcidin, the main negative regulator of iron absorption and recycling, and is mediated by secretion of erythroferrone from bone marrow cells. Targeted treatment for ineffective erythropoiesis is unavailable. Furthermore, molecular mechanisms involved in ineffective erythropoiesis and the details of how erythropoiesis regulates iron metabolism are incompletely understood. Lastly, while loss of erythroferrone in β-thalassemic mice leads to partial reversal of iron overload [Kautz Blood 2015], erythroferrone ablated mice are still able to suppress hepcidin after phlebotomy [Kautz Nat Med 2014]. These finding provide evidence of additional regulatory crosstalk between erythropoiesis and iron metabolism. We hypothesize that bone-marrow derived exosomes regulate iron metabolism by modulating hepcidin. Exosomes are small extracellular vesicles derived from multi-vesicular bodies forming intraluminal vesicles which fuse with the plasma membrane and are released by many different cell types [Thery Nat Rev Immun 2002]. In light of their capacity for cell-cell communication and modification of the microenvironment, exosomes have been widely studied in multiple diseases [Valadi Nat Cell Bio 2007] despite which, erythropoiesis-derived exosomes and their role in iron metabolism regulation remain unexplored. Our preliminary data demonstrate that phlebotomy in wild type mice results in increased exosome concentration in serum and that exosomes are increased in th3/+ mouse serum (Figure 1a). Furthermore, hepcidin induction by exosome depleted-FBS is decreased relative to FBS (Figure 1b), and exosomes isolated from FBS induce hepcidin in a dose response manner in vitro (Figure 1c). We thus propose to explore the mechanistic relationship between exosomes and hepcidin regulation in β-thalassemia. Serum samples from patients with β-thalassemia major and age / gender matched controls were collected; all patients were treated with iron chelation therapy and all samples were collected immediately prior to transfusion. Exosome fractions were purified and analyzed in patients relative to controls. Although there is no difference in the number of exosomes or mean particle size within the exosomal fraction, exosomal protein content per volume of serum is significantly decreased in patients relative to controls. In addition, the treatment of primary wild type mouse hepatocytes with sera from patients and controls reveals the expected relatively decreased hepcidin induction in β-thalassemic patient sera treated hepatocytes relative to control sera; a similar difference is seen in hepatocytes treated with exosome-depleted sera from patients and controls (Figure 2a). These findings suggest that hepcidin suppression is a consequence of the exosome-free portion of serum from control and β-thalassemic samples. Furthermore, only exosomes derived from β-thalassemic patient sera induces hepcidin expression in primary wild type mouse hepatocyte cultures (Figure 2b). Lastly, exosomes derived from β-thalassemic patient sera do not affect ERK1/2 and STAT3 signaling in primary hepatocytes but increase SMAD1/5/8 (Figure 2c) and decrease AKT signaling (Figure 2d). Taken together, these findings demonstrate that exosomes enhance hepcidin expression via increased SMAD1/5/8 signaling, that increased hepcidin may influence multiple signaling pathways by an autocrine mechanism in response to exosomes, and that exosomes counterbalance hepcidin suppressive substances in the exosome-depleted serum from β-thalassemic samples. Our studies provide novel insights into the important previously unexplored mechanism of hepcidin regulation by exosomes in both physiologic and pathologic states. Disclosures Coates: apo pharma: Consultancy, Honoraria, Speakers Bureau; vifor: Consultancy, Honoraria; celgene: Consultancy, Honoraria, Other: steering committee of clinical study; agios pharma: Consultancy, Honoraria. Ginzburg:La Jolla Pharma: Membership on an entity's Board of Directors or advisory committees.

Inhibition of biliary network reconstruction by benzbromarone delays recovery from pre-existing liver injury

The liver performs a variety of essential functions; hence drug-induced liver injury (DILI) is a serious concern that can ultimately lead to the withdrawal of a drug from the market or discontinuation of drug development. However, the mechanisms of drug-induced liver injury are not always clear. We hypothesized that drugs may inhibit the liver recovery process, especially bile canalicular (BC) network reformation, leading to persistent liver injury and deterioration, and tested this hypothesis in the present work. The BC structure disappeared in mice following treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or thioacetamide (TAA) for 4 weeks, then reappeared after 4 weeks of receiving a normal diet. By contrast, reconstruction of the BC structure was suppressed in mice fed a diet containing 0.3% benzbromarone (BBR; which can induce fatal liver injury in clinical settings) after liver injury. Plasma ALT levels were increased significantly in mice treated with BBR after DDC or TAA treatment, compared with BBR alone. To confirm whether BBR has a direct inhibitory effect on hepatocytes, we also examined BC reformation in primary cultured mouse hepatocytes with a sandwich configuration. Under these culture conditions, the BC network rapidly reformed from days 2 and 3 after seeding. During the reformation period, BBR inhibited BC reformation significantly. These results suggest that BBR inhibits BC reconstruction and delays recovery from pre-existing liver injury.

KAPA HiFi HotStart ReadyMix（KK2601）:Validation of the effect of siRNA-mediated knockdown of IFNγ-induced IDO (indoleamine 2,3-dioxygenase) in primary-cultured mouse hepatocytes

KAPA HiFi HotStart ReadyMix（KK2601）:Validation of the effect of siRNA-mediated knockdown of IFNγ-induced IDO (indoleamine 2,3-dioxygenase) in primary-cultured mouse hepatocytes

Strain differences between CD-1 and C57BL/6 mice in expression of metabolic enzymes and DNA methylation modifications of the primary hepatocytes

Primary mouse hepatocyte cultures are widely used in toxicological and pharmacological studies. However, the strain differences in alterations of metabolic enzymes and the regulation of gene expression in response to different stimuli remains unclear. To address this issue, we examined the expression of metabolic enzymes and the regulatory role of DNA methylation in the primary hepatocytes of two mouse strains, CD-1 and C57BL/6. Primary culture of mouse hepatocytes was established using collagen sandwich configuration. Analysis of gene expression of 24 phase I, 18 phase II, and 6 phase III metabolic enzymes on 4 consecutive days after cell seeding revealed that the basal levels of most enzymes in primary cultured hepatocytes differed greatly between the two mouse strains. However, the dynamic changes in most genes were identical between the two strains. In addition, treatment with 3-methylcholanthrene, phenobarbital, and rifampin led to the induction of cytochrome P-450 (cyp) 1a1 and cyp1a2, cyp2b10, cyp3a11. However, induction varied in degree between the two types of primary hepatocytes. The dynamic changes in global DNA methylation and the expression of DNA methylation regulatory factors of the two mouse strains were similar. Of the genes down-regulated over the culture period, hypermethylation of cyp2e1 gene appeared in both mouse strains and led to a suppression of gene expression. Taken together, these results demonstrate that the expression of metabolic enzymes and the response to agonists in primary hepatocytes differ between CD-1 and C57BL/6 mouse strains. Epigenetic regulation might be involved in the suppression of cyp 450s’ expression.

Optimization of DamID for use in primary cultures of mouse hepatocytes.

DamID, a method to identify DNA associating with a particular protein, was originally developed for use in immortalized tissue culture lines. The power of this technique has led to its adaptation for a number of additional systems. Here we report adaptations for its use in primary cells isolated from rodents with emphasis on the challenges this presents. Specifically, we present several modifications that allow the method to be performed in mouse acutely isolated primary hepatocytes while seemingly maintaining tissue genome architecture. We also describe the downstream bioinformatic analysis necessary to identify LADs and discuss some of the parameters and their effects with regards to the sensitivity of the method.

Co-administration of nuciferine reduces the concentration of metformin in liver via differential inhibition of hepatic drug transporter OCT1 and MATE1.

Nuciferine (NF), one of the main and effective components in Nelumbo nucifera Gaertn. leaf extracts, is a promising drug candidate for the treatment of obesity-related diseases, while metformin is a first line therapeutic drug for type 2 diabetes mellitus. Since nuciferine and metformin are likely to be co-administered, the aim of the present study was to evaluate whether co-administration of nuciferine would influence the liver (target tissue) distribution and the anti-diabetic effect of metformin by inhibiting hepatic organic cation transporter 1 (OCT1) and multidrug and toxin extrusion 1 (MATE1). The data demonstrated that nuciferine significantly reduced metformin accumulation in MDCK cells stably expressing human OCT1 (MDCK-hOCT1) or hMATE1 (MDCK-hMATE1), and primary cultured mouse hepatocytes. Furthermore, the presence of nuciferine in the basal compartment caused a concentration-dependent reduction of intracellular metformin accumulation in MDCK-hOCT1/hMATE1 cell monolayers. Compared with the metformin treatment-alone group, co-administration of nuciferine (40mg/kg) markedly reduced the metformin concentration in mouse livers at 30 and 60min after a single oral dose of metformin (200mg/kg), and subsequently impaired the glucose-lowering effect of metformin (200mg/kg), but the glucose-lowering effect became no different at 90 and 120min. Therefore, nuciferine influenced the liver concentration and glucose-lowering effect of metformin only for a period of time after dose, administration of nuciferine and metformin with an interval might prevent the drug-drug interaction mediated by OCT1 and MATE1.