AML12 Cell Line Research Articles

Assessing the role of surface glycans of extracellular vesicles on cellular uptake

Extracellular vesicles (EVs) are important mediators of cell-cell communication in a broad variety of physiological contexts. However, there is ambiguity around the fundamental mechanisms by which these effects are transduced, particularly in relation to their uptake by recipient cells. Multiple modes of cellular entry have been suggested and we have further explored the role of glycans as potential determinants of uptake, using EVs from the murine hepatic cell lines AML12 and MLP29 as independent yet comparable models. Lectin microarray technology was employed to define the surface glycosylation patterns of EVs. Glycosidases PNGase F and neuraminidase which cleave N-glycans and terminal sialic acids, respectively, were used to analyze the relevance of these modifications to EV surface glycans on the uptake of fluorescently labelled EVs by a panel of cells representing a variety of tissues. Flow cytometry revealed an increase in affinity for EVs modified by both glycosidase treatments. High-content screening exhibited a broader range of responses with different cell types preferring different vesicle glycosylation states. We also found differences in vesicle charge after treatment with glycosidases. We conclude that glycans are key players in the tuning of EV uptake, through charge-based effects, direct glycan recognition or both, supporting glycoengineering as a toolkit for therapy development.

Adenine nucleotide-mediated regulation of hepatic PTP1B activity in mouse models of type 2 diabetes.

Plasma 5'-AMP (pAMP) is elevated in mouse models of type 2 diabetes. However, the metabolic regulatory role of adenine nucleotides in type 2 diabetes remains unclear. Adenine nucleotides and their metabolites in plasma and liver were examined by HPLC. 1H NMR-based metabolomics analysis was performed to explore the changes of metabolites in mouse models of type 2 diabetes. Na+/K+ ATPase and Na+/H+ exchanger activity were measured in response to adenine nucleotide metabolites. Human recombinant protein tyrosine phosphatase 1B (PTP1B) was used for enzyme kinetic assays. Protein binding assays were performed with microscale thermophoresis. The intracellular pH of hepatocyte AML12 cell lines was measured using the BCECF-AM method. We also analysed pAMP levels in participants with type 2 diabetes. Elevation of pAMP was a universal phenomenon in all mouse models of type 2 diabetes including db/db vs lean mice (13.9 ± 2.3μmol/l vs 3.7 ± 0.9μmol/l; p < 0.01), ob/ob vs lean mice (9.1 ± 2.0μmol/l vs 3.9 ± 1.2μmol/l; p < 0.01) and high-fat diet/streptozotocin-induced vs wild-type mice (6.6 ± 1.5μmol/l vs 4.1 ± 0.9μmol/l; p < 0.05); this elevation was required for the occurrence of hyperglycaemia in obese mice. 1H NMR-based metabolomics study following HPLC analysis revealed that the metabolite profile in wild-type mice treated with 5'-AMP was similar to that in db/db diabetic mice, especially the accumulation of a large quantity of ATP and its metabolites. The glucose-lowering drug metformin reduced the severity of hyperglycaemia both in 5'-AMP-induced wild-type mice and db/db mice. Metformin decreased the accumulation of liver ATP but not its metabolites in these hyperglycaemic mice. ATP and metformin reciprocally change cellular pH homeostasis in liver, causing opposite shifts in liver activity of PTP1B, a key negative regulator of insulin signalling. Furthermore, pAMP levels were also elevated in individuals with type 2 diabetes (45.2 ± 22.7nmol/l vs 3.1 ± 1.9nmol/l; p < 0.01). These results reveal an emerging role for adenine nucleotide in the regulation of hyperglycaemia and provide a potential therapeutic target in obesity and type 2 diabetes.

Propofol alleviates oxidative stress via upregulating lncRNA-TUG1/Brg1 pathway in hypoxia/reoxygenation hepatic cells.

Reducing oxidative stress is an effective method to prevent hepatic ischaemia/reperfusion injury (HIRI). This study focuses on the role of propofol on the oxidative stress of hepatic cells and the involved lncRNA-TUG1/Brahma-related gene 1 (Brg1) pathway in HIRI mice. The mouse HIRI model was established and was intraperitoneally injected with propofol postconditioning. Hepatic injury indexes were used to evaluate HIRI. The oxidative stress was indicated by increasing 8-isoprostane concentration. Mouse hepatic cell line AML12 was treated with hypoxia and subsequent reoxygenation (H/R). The targeted regulation of lncRNA-TUG1 on Brg1 was proved by RNA pull-down, RIP (RNA-binding protein immunoprecipitation) and the expression level of Brg1 responds to silencing or overexpression of lncRNA-TUG1. Propofol alleviates HIRI and induces the upregulation of lncRNA-TUG1 in the mouse HIRI model. Propofol increases cell viability and lncRNA-TUG1 expression level in H/R-treated hepatic cells. In H/R plus propofol-treated hepatic cells, lncRNA-TUG1 silencing reduces cell viability and increased oxidative stress. LncRNA-TUG1 interacts with Brg1 protein and keeps its level via inhibiting its degradation. Brg1 overexpression reverses lncRNA-TUG1 induced the reduction of cell viability and the increase in oxidative stress. LncRNA-TUG1 silencing abrogates the protective role of propofol against HIRI in the mouse HIRI model. LncRNA-TUG1 has a targeted regulation of Brg1, and thereby affects the oxidative stress induced by HIRI. This pathway mediates the protective effect of propofol against HIRI of hepatic cell.

Abstract 985: Development and validation of cell-based TFEB translocation assay in a high-content and high-throughput screening format

Abstract Transcription factor EB (TFEB) is a master regulator of lysosomal gene expression and mitochondrial biogenesis. Autophagy is known to act as a double edged sword in cancer. Autophagy acts as a tumor suppressor during the tumorigenesis process but it also helps to sustain the survival of already transformed cancer cells. Lysosomes sit at the end of the autophagy pathway, which is critical to meet the needs of autophagy. Therefore, small molecules that inhibit TFEB-mediated lysosomal biogenesis and autophagic degradation may be helpful for inhibiting cancer cell growth and limiting cancer progression. On the other hand, small molecules that enhance TFEB-mediated lysosomal degradation may be used to prevent tumorigenesis. In order to identify compounds that modulate TFEB translocation, an image-based TFEB translocation assay was developed in a 1536-well plate format with a GFP-TFEB stable AML12 cell line, which is an immortalized mouse hepatocyte cell line. Torin 1, a known TFEB translocation inducer, was used as positive control in the study. Torin 1 increased TFEB nuclear translocation in a concentration dependent manner with an EC50 of 150nM. To optimize the assay condition, various cell densities per well and time courses were tested. To validate this assay, a group of 384 known anticancer compounds were screened at 11 concentrations ranging from 0.8 nM to 46 μM. From this screening, we identified 25 compounds that enhanced TFEB nuclear translocation with EC50s ranging from 2 nM to 12 μM. Several known and novel TFEB inducers were among these compounds. These results demonstrate that this high-content and high-throughput assay can be used to screen large numbers of chemicals and provide a robust in vitro system to identify TFEB agonists and antagonists, which may be used to either prevent (agonists) or treat (antagonist) cancer. Citation Format: Li Zhang, Ruili Huang, Wen Xing Ding, Menghang Xia. Development and validation of cell-based TFEB translocation assay in a high-content and high-throughput screening format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 985.

1872-P: Hepatic miR-582-5p Is Programmed by Maternal Diet-Induced Obesity in Male Mice and Contributes to Nonalcoholic Fatty Liver Disease

Obesity during pregnancy is associated with increased risk of nonalcoholic fatty liver disease (NAFLD) in the offspring; however, the underlying molecular mechanisms remain largely unknown. The aim of the current study was to explore the contribution of microRNAs (miRs) as mediators of programming by maternal obesity. To achieve this, we used a mouse model of maternal-diet induced obesity where dams develop gestational diabetes and offspring demonstrate increased hepatic triglyceride content. We carried out small RNA next generation sequencing analysis of liver from twelve-week-old male offspring and identified miR-582-5p as the most dysregulated, showing a threefold increase. Binding site prediction (TargetScan 7.2) and pathway enrichment analysis (Reactome v67) suggested a number of miR-582-5p target genes related to lipid metabolism including Insulin Induced Gene 2 (INSIG2) and Translocase of Inner Mitochondrial Membrane 23 (TIMM23). Overexpression of miR-582-5p in AML12 (mouse) and HEPG2 (human) cell lines caused increased triglyceride accumulation, downregulation of INSIG2 and TIMM23, increased nuclear localization of Sterol Regulatory Element-Binding Protein 1 (SREBP-1), and overexpression of lipogenic genes. These findings suggest that overexpression of miR-582-5p induces hepatic intracellular lipid accumulation through mechanisms that involve mitochondrial malfunction and activation of de novo lipogenesis. We therefore conclude that an epigenetically programmed increase in hepatic miR-582 is a likely contributing mechanism underlying the programming of fatty liver disease in the offspring of obese gestational diabetic mothers. Disclosure L. Pantaleao: None. D. Fernandez-Twinn: None. A. Carpenter: None. E. Loche: None. L. Dearden: None. D. Duque Guimaraes: None. N.C. Penfold: None. T.J. Ashmore: None. M. Bushell: None. S. Ozanne: Advisory Panel; Self; Nestlé. Funding Brazilian National Council for the Improvement of Higher Education (BEX 10594/13-2); Brazilian National Council for Scientific and Technological Development (PDE/204416/2014-0)

The mir-465 family is upregulated with age and attenuates growth hormone signaling in mouse liver.

We analyzed the small RNA transcriptome from 5‐month‐old, 24‐month‐old, and 36‐month‐old mouse liver and found 56 miRNAs that changed their expression profile with age. Among these is a cluster of 18 miRNAs that are upregulated between 50‐ and 1,000‐fold at 24 and 36 months of age. This cluster is located in a 60‐kb region of the X‐chromosome that is devoid of other coding sequences and is part of a lamin‐associated domain. Potential targets of the miRNAs in the cluster suggest they may regulate several pathways altered in aging, including the PI3K‐Akt pathway. Total transcriptome analyses indicate that expression of several potential genes in the PI3K‐Akt pathway that may be targeted by the mir‐465 family (mmu‐mir‐465a, mmu‐mir‐465b, and mmu‐mir‐465c) is downregulated with age. Transfection of the liver cell line AML12 with mir‐465 family members leads to a reduction of three of these potential targets at the mRNA level: a 40% reduction of the growth hormone receptor (GHR), and a 25% reduction in Kitl and PPP2R3C. Further investigation of the GHR 3′UTR revealed that the mir‐465 family directly targets the GHR mRNA. Cells transfected with mir‐465 showed a reduction of JAK2 and STAT5 phosphorylation upon growth hormone (GH) stimulation, resulting in a reduction in insulin‐like growth factor 1 (IGF‐1) and IGF‐1‐binding protein 3 expression. With age, GH signaling falls and there is a reduction in circulating IGF‐1. Our data suggest that an increase in expression of the mir‐465 family with age may contribute to the reduction in the GH signaling.

Suppression of YAP/TAZ-Notch1-NICD axis by bromodomain and extraterminal protein inhibition impairs liver regeneration.

Background and aims: Biological mechanisms that control liver regeneration remain poorly defined. However, these mechanisms are remarkable issues in the clinic that affect management of hepatic loss caused by liver surgery, traumatic injury, chronic infection, or liver poisoning. Increasing evidence has shown that various growth factors, cytokines, and metabolic signaling pathways affect the liver regenerative process. Our aim is to study the effect of bromodomain and extraterminal (BET) protein inhibition on liver regeneration and its mechanism.Methods: We studied the role of BET protein inhibitor, JQ1, in liver regeneration in a mouse model after 70% partial hepatectomy (PH). We evaluated yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) and Notch signaling pathways, which were affected by BET protein inhibitor in mouse hepatic tissues and primary hepatocytes in vivo and AML12 cell lines in vitro. We evaluated the relationship of YAP/TAZ and Notch signaling pathway using YAP/TAZ pathway inhibitor in liver regeneration in vivo. Moreover, we analyzed the relationship of YAP/TAZ and Notch signaling pathways via overexpression or RNA silencing of Yap in AML12 cells. Furthermore, we used Yap overexpression mouse model to examine whether it can rescue liver regeneration damage caused by inhibition of BET proteins.Results: In this study, we report that BET protein inhibitor JQ1 molecule impairs the early phase of liver regeneration in a mouse model after 70% PH. Mechanistically, YAP/TAZ and Notch1-NICD pathways were suppressed by BET protein inhibitor in mouse hepatic tissues and primary hepatocytes in vivo and mouse AML12 cell lines in vitro. By using YAP/TAZ pathway inhibitor, we confirmed that the liver regeneration and the activation of Notch pathway were impaired by the inhibition of YAP/TAZ pathway in vivo. Furthermore, the study showed that Yap knockdown by shRNA in normal mouse hepatic cell line downregulated Notch1 signal transduction, whereas Yap overexpression promoted Notch1-NICD signals. Specific overexpression of Yap in mouse liver could rescue the effect of BET protein inhibition on liver regeneration injury.Conclusion: These results revealed the crucial role of the YAP/TAZ-Notch1-NICD axis in liver regeneration. Therefore, BET protein inhibitors must be used in caution in the treatment of hepatic diseases by reason of its suppressive roles in liver regeneration.

Robust Human and Murine Hepatocyte Culture Models of Hepatitis B Virus Infection and Replication.

Hepatitis B virus (HBV) is a major cause of chronic liver diseases, including hepatitis, cirrhosis, and hepatocellular carcinoma. HBV research has been hampered by the lack of robust cell culture and small animal models of HBV infection. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor has been a landmark advance in HBV research in recent years. Ectopic expression of NTCP in nonpermissive HepG2, Huh7, and AML12 cell lines confers HBV susceptibility. However, HBV replication in these human and murine hepatocyte cell lines appeared suboptimal. In the present study, we constructed stable NTCP-expressing HepG2 and AML12 cell lines and found that HBV permissiveness is correlated with NTCP expression. More significantly, we developed robust HBV cell culture models by treating the HBV-infected cells with dimethyl sulfoxide (DMSO) and hydrocortisone, which significantly promoted HBV replication and production. Mechanistic studies suggested that hydrocortisone significantly enhanced the transcription and expression of PGC1α and HNF4α, which are known to promote HBV transcription and replication. These new human and murine hepatocyte culture systems of HBV infection and replication will accelerate the determination of molecular aspects underlying HBV infection, replication, and morphogenesis in human and murine hepatocytes. We anticipate that our HBV cell culture models will also facilitate the discovery and development of antiviral drugs towards the ultimate eradication of chronic hepatitis B virus infection.IMPORTANCE HBV research has been greatly hampered by the lack of robust cell culture and small animal models of HBV infection and propagation. The discovery of NTCP as an HBV receptor has greatly impacted the field of HBV research. Although HBV infection of NTCP-expressing human and murine hepatocyte cell lines has been demonstrated, its replication in cell culture appeared inefficient. To further improve cell culture systems of HBV infection and replication, we constructed NTCP-expressing HepG2 and AML12 cell lines that are highly permissive to HBV infection. More significantly, we found that DMSO and hydrocortisone markedly enhanced HBV transcription and replication in human and murine hepatocytes when added to the cell culture medium. These new cell culture models of HBV infection and replication will facilitate HBV research and antiviral drug discovery towards the ultimate elimination of chronic hepatitis B virus infection.

Suitability of hepatocyte cell lines HepG2, AML12 and THLE-2 for investigation of insulin signalling and hepatokine gene expression.

Immortal hepatocyte cell lines are widely used to elucidate insulin-dependent signalling pathways and regulation of hepatic metabolism, although the often tumorigenic origin might not represent the metabolic state of healthy hepatocytes. We aimed to investigate if murine cell line AML12 and human cell line THLE-2, which are derived from healthy liver cells, are comparable to hepatoma cell line HepG2 for studying acute insulin signalling and expression of gluconeogenic enzymes and hepatokines. Insulin responsiveness of AML12 and THLE-2 cells was impaired when cells were cultured in the recommended growth medium, but comparable with HepG2 cells by using insulin-deficient medium. THLE-2 cells showed low abundance of insulin receptor, while protein levels in HepG2 and AML12 were comparable. AML12 and THLE-2 cells showed only low or non-detectable transcript levels of G6PC and PCK1. Expression of ANGPTL4 was regulated similarly in HepG2 and AML12 cells upon peroxisome proliferator-activated receptor δ activation but only HepG2 cells resemble the in vivo regulation of hepatic ANGPTL4 by cAMP. Composition of the culture medium and protein expression levels of key signalling proteins should be considered when AML12 and THLE-2 are used to study insulin signalling. With regard to gluconeogenesis and hepatokine expression, HepG2 cells appear to be closer to the in vivo situation despite the tumorigenic origin.

Effect of miR-433-3p and miR-883b-5p on murine CYP 3A family enzymes in AML12 cells.

Here we searched for microRNAs that could interact with cytochrome P450 (CYP) enzymes in silico, and then investigated their effects on Cyp gene expressions using the cultured mouse liver cell line AML12. Among the mouse Cyp3a genes, some miRNAs were found to interact with Cyp3a11, 13, 16, and 44 by the in silico analysis using the miRWalk2.0 database. In addition to this software, which included twelve miRNA target prediction algorithms, we also applied our in-house-developed Excel VBA algorithm to obtain predictions more efficiently. Finally, two miRNAs, miR-433-3p and miR-883b-5p, were extracted as candidates that interact with Cyp3a genes. To evaluate the effects of these miRNAs on Cyp3a gene expression, we first examined whether they actually interacted with the Cyp3a 3'-untranslated region (3'-UTR) using a luciferase assay system in AML12 cells. We then evaluated whether the expression of each miRNA affected the expression of Cyp3a mRNAs and their transcribed proteins. We found that the transiently expressed miRNAs significantly reduced the reporter activity of the Cyp3a 3'-UTR site in AML12 cells. In addition, the mRNA and protein expressions of the corresponding Cyp3as were significantly decreased in the miRNA-treated AML12 cells. Using cultured cells, we clearly demonstrated that miR-433-3p and miR-883b-5p, which were identified by in silico prediction, actually bind to Cyp3a mRNAs and regulate Cyp gene expressions.

Luteolin induces caspase-dependent apoptosis via inhibiting the AKT/osteopontin pathway in human hepatocellular carcinoma SK-Hep-1 cells

AimsLuteolin, a naturally occurring flavonoid, possesses anti-cancer effects including induction of apoptosis. This study investigated the involvement of osteopontin (OPN) in luteolin-induced apoptosis in human hepatocellular carcinoma (HCC) SK-Hep-1 cells with high OPN expression. Main methodsMTT assay was used to determine the cell viability. Cell cycle analysis was performed to identify apoptosis. Apoptosis was confirmed by detecting cytoplasmic histone-associated-DNA-fragments using a cell death detection ELISAPLUS kit. The expression of proteins was evaluated by Western blot. Reverse transcriptase-polymerase chain reaction was employed to detect the expression of mRNA. Key findingsCytotoxic effect of luteolin was higher in cancer cell line SK-Hep-1 cells than in normal cell line AML12 cells. Luteolin led a significantly increase in apoptosis accompanied by activation of caspase 8, −9 and −3 and cleavage of poly (ADP-ribose) polymerase (PARP), which was completely blocked by Z-VAD-FMK, a pan caspase inhibitor. Luteolin significantly downregulated the expression of X-linked inhibitor of apoptosis (XIAP), Mcl-1 and Bid. Furthermore, luteolin effectively decreased OPN expression at both mRNA and protein level. Exogenous OPN markedly blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of XIAP and Mcl-1 in luteolin-treated cells. Luteolin impaired the AKT pathway by inhibiting the phosphorylation of AKT. SC79, an AKT activator, blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of OPN, XIAP, Mcl-1 and Bid in luteolin-treated cells. SignificanceThese results demonstrated that luteolin inhibits the AKT/OPN pathway, thereby inducing caspase-dependent apoptosis in human HCC SK-Hep-1 cells with little toxicity.

CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase

ALDH1L1 is a folate-metabolizing enzyme abundant in liver and several other tissues. In human cancers and cell lines derived from malignant tumors, the ALDH1L1 gene is commonly silenced through the promoter methylation. It was suggested that ALDH1L1 limits proliferation capacity of the cell and thus functions as putative tumor suppressor. In contrast to cancer cells, mouse cell lines NIH3T3 and AML12 do express the ALDH1L1 protein. In the present study, we show that the levels of ALDH1L1 in these cell lines fluctuate throughout the cell cycle. During S-phase, ALDH1L1 is markedly down regulated at the protein level. As the cell cultures become confluent and cells experience increased contact inhibition, ALDH1L1 accumulates in the cells. In agreement with this finding, NIH3T3 cells arrested in G1/S-phase by a thymidine block completely lose the ALDH1L1 protein. Treatment with the proteasome inhibitor MG-132 prevents such loss in proliferating NIH3T3 cells, suggesting the proteasomal degradation of the ALDH1L1 protein. The co-localization of ALDH1L1 with proteasomes, demonstrated by confocal microscopy, supports this mechanism. We further show that ALDH1L1 interacts with the chaperone-dependent E3 ligase CHIP, which plays a key role in the ALDH1L1 ubiquitination and degradation. In NIH3T3 cells, silencing of CHIP by siRNA halts, while transient expression of CHIP promotes, the ALDH1L1 loss. The downregulation of ALDH1L1 is associated with the accumulation of the ALDH1L1 substrate 10-formyltetrahydrofolate, which is required for de novo purine biosynthesis, a key pathway activated in S-phase. Overall, our data indicate that CHIP-mediated proteasomal degradation of ALDH1L1 facilitates cellular proliferation.

Lycium barbarum Polysaccharide Supplementation Improves Alcoholic Liver Injury in Female Mice by Inhibiting Stearoyl-CoA Desaturase 1.

Lycium barbarum polysaccharide (LBP) is a water fraction of wolfberry, which has been demonstrated to possess a hepatoprotective effect in several liver disease models. However, the anti-alcoholic liver disease (anti-ALD) mechanism of LBP has not been investigated thoroughly. Its protective effects on both male and femal mice are investigated in the current study. A chronic ethanol-fed ALD in vivo model is applied to study the effect of LBP in both male and female mice. It is observed that ethanol causes more severe liver injury in female than male mice, and the ameliorative effects of LBP are also more significant in female mice, which are impaired after complete bilateral oophorectomy. The hepatic SCD1 expression is found to be positively correlated with the severity of the liver damage and the main mediator of LBP inducer of protection. The AMPK-CPT pathway is also activated by LBP to rebalance the dysregulated lipid metabolism during ALD development. By using concurrent sodium palmitate and an ethanol-induced in vitro cell damage model in AML-12 cell line, it is characterized that LBP directly interacts with ERα instead of ERβ to activate the SCD1-AMPK-CPT pathway. LBP is an effective and safe hepatoprotective agent against ALD primarily through the SCD1-AMPK-CPT pathway after ERα agonist.

Hepatocyte-specific deletion of LASS2 protects against diet-induced hepatic steatosis and insulin resistance.

Homo sapienslongevity assurance homolog 2 of yeast LAG1 (LASS2) is expressed mostly in human liver. Here, we explored roles of LASS2 in pathogenesis of hepatic steatosis. Hepatocyte-specific LASS2 knockout (LASS2-/-) mice were generated using Cre-LoxP system. LASS2-/- and wild-type (WT) mice were fed with chow or high-fat diet (HFD). We found LASS2-/- mice were resistant to HFD-induced hepatic steatosis and insulin resistance. In HFD-fed mice, LASS2 deficiency significantly inhibited p38 MAPK and ERK1/ERK2 signaling in mouse liver. This effect was mediated by a significant increase of V-ATPase activity and a decrease of ROS level. We also observed that elevated expression of LASS2 in mouse hepatocyte cell line AML12 obviously decreased V-ATPase activity and increased ROS level by activation of p38 MAPK and ERK1/ERK2 signaling. Our findings indicate that LASS2 plays an important role in the pathogenesis of diet-induced hepatic steatosis and is a potential novel target for prevention and intervention of liver diseases.

MiR-203 Inhibits Alcohol-Induced Hepatic Steatosis by Targeting Lipin1.

Alcoholic liver disease (ALD) is a global liver disease which characterized by liver inflammation, fatty liver, alcoholic hepatitis, or liver cirrhosis. Alcohol abuse is one of the main reasons for liver disease. Alcoholic fatty liver (AFL) disease is the early stage of ALD and associated with the excessive lipids accumulation in hepatocytes as well as oxidative stress. MicroRNA-203 (miR-203) is known to suppress the proliferation and metastasis of hepatocellular carcinoma, but the role in the progression of alcoholic liver disease is not clear and is warranted for further investigation. In the present study, we have found the expression of miR-203 is down-regulated in Gao-Binge alcoholic mice model and ethanol-induced AML-12 cell lines in vitro. Furthermore, over-expression of miR-203 decrease the lipids accumulation in liver and ethanol-induced AML-12 cells. Mechanistically, we identified that Lipin1 is a key regulator of hepatic lipid metabolism, and acts as a downstream target for miR-203. In summary, our results suggested that over-expression of miR-203 inhibited the liver lipids accumulation and the progression of AFL by targeting Lipin1.

Caffeic acid ameliorates hepatic steatosis and reduces ER stress in high fat diet-induced obese mice by regulating autophagy.

Non-alcoholic fatty liver disease is characterized by high hepatic triacylglycerol contents, which is associated with endoplasmic reticulum (ER) stress and insulin resistance. Caffeic acid (CA) has antioxidant, immunomodulatory, and antiinflammatory effects. We investigated the effects of CA on hepatic steatosis and its mechanism of action. We treated CA (50 µM) with AML12 cells. We categorized mice into three groups as follows: low-fat diet mice (LFD, n = 10), high-fat diet-induced obese mice (HFD, n = 10), and HFD fed with CA (50 mg/kg/d, n = 10) for 10 wk. CA did not cause any cytotoxic effect on AML12 cell line within the range of concentrations tested (0-200 µM). We found that CA (50 µM) treatment in palmitate-treated AML12 hepatocytes reduced lipid accumulation and lipogenesis markers, decreased ER stress, and increased autophagy markers. However, there was no significant difference in lipid droplets of palmitate-treated AML12 hepatocytes and CA-treated autophagy-related protein 7 deficiency AML12 hepatocytes with palmitate. Similarly, CA significantly lowered body and liver weights. Lipid accumulation in the liver decreased in the HFD + CA group compared with the HFD group. Glucose intolerance and insulin sensitivity also were markedly improved in the HFD + CA group. Moreover, the levels of ER stress markers were decreased in the livers of the HFD + CA group. Autophagy markers were increased in the livers of the HFD + CA group. These results suggest that caffeic acid may ameliorate hepatic steatosis and decrease ER stress by increasing autophagy.

Taurocholate Induces Connective Tissue Growth Factor Expression in Hepatocytes Through ERK-YAP Signaling

Background/Aims: Cholestasis is characterized by intrahepatic accumulation of cytotoxic bile acids (BAs), ultimately leading to fibrosis and cirrhosis, but the precise role of BAs in cholestasis-induced liver fibrosis remains largely elusive. In this study, we investigated the role and the potential mechanisms of BAs during cholestasis in vivo and in vitro. Methods: The effect of BAs during cholestasis was studied in bile duct ligation (BDL) rat models in vivo. We performed immunohistochemistry, Western blotting, and quantitative RT-PCR to investigate the expression of connective tissue growth factor (CTGF/CCN2) in rat liver during cholestasis. The hepatic cell lines AML12 and BRL were stimulated with taurocholate (TC) and the level of CTGF/CCN2, and activation of ERK, Akt, p38 MAPK, JNK, YAP, and TGF-β/Smad signaling were examined using Western blotting. Next, to elucidate the mechanism underlying bile acid-induced CTGF/CCN2, we treated the cells with MEK1/2 inhibitor (U0126), YAP function inhibitor (verteporfin), p38 kinase inhibitor (SB203580), Akt inhibitor (MK2206), and small interfering RNA (siRNA) targeting mek1, erk, and yap in cooperation with TC. Besides, we confirmed the activation of these signaling pathways in BDL and sham rat livers by immunohistochemistry, Western blotting, and quantitative RT-PCR. Results: In this study, we confirmed that the expression of CTGF/CCN2 was increased in BDL-induced rodent cholestatic liver fibrosis. In addition, we showed that TC, the main component of BAs, enhanced the synthesis of CTGF/ CCN2 in AML12 and BRL hepatic cell lines. Moreover, we demonstrated that TC activated ERK, Akt, and YAP signaling in hepatocytes, but the precise roles of these signaling cascades in the expression of CTGF/CCN2 were different: TC-induced expression of CTGF/CCN2 was mediated by ERK-YAP signaling, whereas Akt signaling inhibited ERK signaling and YAP and subsequently the expression of CTGF/CCN2 in hepatocytes. Furthermore, YAP functioned as a downstream regulator of ERK signaling in TC-induced CTGF/CCN2 expression in hepatocytes. Conclusion: Our report provides evidence for the role of conjugated BAs in liver fibrosis and suggests that the production of CTGF/CCN2, induced by conjugated BAs via ERK-YAP axis activation, may be a therapeutic target in cholestasis-induced liver fibrosis.

SIRT3 protects hepatocytes from oxidative injury by enhancing ROS scavenging and mitochondrial integrity

Evidences of oxidative stress and mitochondrial dysfunction have been recognized in most of clinical and experimental liver diseases. SIRT3, a member of NAD+-dependent deacetylases, is mainly localized in mitochondria. So far, the role of SIRT3 in protecting hepatocytes against oxidative stress remains elusive. Herein, we found SIRT3 protein expression is decreased in tert-butyl hydroperoxide (t-BHP)-treated AML12 cells in vitro and primary hepatocytes from CCl4-injured mice in vivo. To further verify the role of SIRT3 in protecting hepatocytes from t-BHP-induced injury, SIRT3 overexpressed AML12 cell line and primary hepatocytes were generated. SIRT3 overexpressed hepatocytes showed improved cell viability upon t-BHP challenge, with less intracellular reactive oxygen species (ROS) accumulation. SIRT3 overexpression reduced superoxide dismutase 2 acetylation level and stimulated nuclear factor erythroid 2-related factor 2 nuclear translocation to enhance anti-oxidative capacity. Moreover, SIRT3 deacetylated peroxisome proliferator-activated receptor γ coactivator 1α to promote mitochondrial biogenesis, and 8-oxoguanine DNA glycosylase 1 to orchestrate DNA repair, resulting in improved mitochondrial function. Through deacetylating Ku70, SIRT3 also abated mitochondrial translocation of dynamin-related protein 1, to attenuate mitochondrial fragmentation in t-BHP-injured hepatocytes. These results suggested that SIRT3 protected hepatocytes against oxidative stress by enhancing ROS scavenging and maintaining mitochondrial integrity.

Aberrant expression of miR-451a contributes to 1,2-dichloroethane-induced hepatic glycerol gluconeogenesis disorder by inhibiting glycerol kinase expression in NIH Swiss mice.

The identification of aberrant microRNA (miRNA) expression during chemical-induced hepatic dysfunction will lead to a better understanding of the substantial role of miRNAs in liver diseases. 1,2-Dichloroethane (1,2-DCE), a chlorinated organic toxicant, can lead to hepatic abnormalities in occupationally exposed populations. To explore whether aberrant miRNA expression is involved in liver abnormalities mediated by 1,2-DCE exposure, we examined alterations in miRNA expression patterns in the livers of NIH Swiss mice after dynamic inhalation exposure to 350 or 700 mg m-3 1,2-DCE for 28 days. Using a microarray chip, we discovered that only mmumiR-451a was significantly upregulated in the liver tissue of mice exposed to 700 mg m-3 1,2-DCE; this finding was validated by quantitative real-time polymerase chain reaction. In vitro study revealed that it was metabolite 2-chloroacetic acid, not 1,2-DCE that resulted in the upregulation of mmu-miR-451a in the mouse AML12 cell line. Furthermore, our data showed that the upregulation of mmu-miR-451a induced by 2-chloroacetic acid could suppress the expression of glycerol kinase and lead to the inhibition of glycerol gluconeogenesis in mouse liver tissue and AML12 cells. These observations provide evidence that hepatic mmu-miR-451a responds to 1,2-DCE exposure and might induce glucose metabolism disorders by suppressing the glycerol gluconeogenesis process.

Construction of all-in-one CRISPR/Cas9 vector system targeting miR-101a gene in mouse hepatic cell line AML12

To develop an all-in-one CRISPR/Cas9 vector system that can efficiently knockdown miR-101a expression in mice. Three sgRNAs targeting mouse miR-101a gene and a small guide (sgRNA) targeting green fluorescent protein gene were designed and constructed into an all-in-one vector system (pENTRY-U6-sgRNA-WT Cas9). Moreover, sgRNA1 and sgRNA3 were selected and constructed into a double-nicking Cas9 vector (pENTRY-U6-sgRNA-U6-sgRNA-Cas9 D10A). The constructed plasmids were transfected into mouse liver AML12 cells for validation by T7 EndoⅠ(T7EⅠ) 72 h after transfection. The pAD vectors were cloned via the Gateway system, and the recombinant adenovirus vectors were packaged in 293A cells. The virus particles were used to infect AML12 cells and the expression levels of mature miR-101a were analyzed to monitor the knockout efficiency after 72 h. The constructed pENTRY all-in-one vectors were validated by gene sequencing and T7EⅠ assay, which showed CRISPR/Cas9-mediated mismatches at target sites of miR-101a gene. The adenovirus vectors were constructed successfully. The CRISPR/Cas9 containing adenovirus was introduced to AML12 cells and the quantitative real-time PCR assays indicated that the expression level of mature miR-101a was significantly decreased compared with that of the control (all P<0.01). We have successfully constructed two "all-in-one" CRISPR/Cas9 vector systems targeting miR-101a gene in mouse liver AML12 cells with high efficiency. It provides experimental basis for research of microRNA, and a reference method for knockout of other miRNAs.