Impact of Variant Notch Ligands on Chronic Liver Diseases

Introduction To date, the roles of individual Notch ligands in liver injury are not well defined. The current study investigated whether and how Notch ligands DLL4 and JAG1 affect liver injury. Methods We examined the expression of Notch ligands and receptors by immunohistochemistry (IHC) in the liver samples of 26 patients with HBV-induced liver cirrhosis. The function of recombinant Dll4 (rDll4) and rJag-1 was investigated in vivo in carbon tetrachloride (CCl4) and bile duct ligation (BDL) animal models and in vitro in hepatocytes, Kupffer cells (KCs), and hepatic stellate cells (HSCs). Results DLL4 and JAG1 were the only Notch ligands expressed in liver sinusoids of examined patients. In the CCl4 animal model, rDll4 and rJag-1 ameliorated liver fibrosis, decreased infiltration of inflammatory cells, and inhibited apoptosis. On the contrary, rDll4 and rJag-1 caused rapid death of all BDL mice within 1 week. rDll4 inhibited the expression of chemokine ligand 2 (CCL2) and infiltration of inflammatory cells in livers of BDL mice, whereas rJag-1 did not have any impact on inflammatory cells infiltration and CCL2 expression. In macrophages and HSCs, rDll4 inhibited LPS-induced CCL2 expression, whereas rJag-1 did not impact CCL2 expression. Inhibition of inflammation by rDll4 caused unrestricted bile infarct and rapid death of BDL animals. Recombinant Ccl2 (rCcl2) restored the infiltration of inflammatory cells, decreased the size of bile infarcts and rescued rDll4-treated BDL animals from death. In ACLF patients, DLL4 expression was negatively associated with expression of CCL2. In contrast to rDll4-treated BDL animals, rCcl2 did not rescue rJag-1-treated BDL animals. Conclusion Etiology determines the effects of DLL4 and JAG1 on liver injury. DLL4 inhibits liver inflammation through inhibiting CCL2, a key chemokine for recruitment of inflammatory cells. How Jag-1 impact liver injury requires further investigation

Mitochondrial respiration is decreased in visceral but not subcutaneous adipose tissue in obese individuals with fatty liver disease

Adipose tissue (commonly called body fat) can be found under the skin (subcutaneous) or around internal organs (visceral). Dysfunction of adipose tissue can cause insulin resistance and lead to excess delivery of fat to other organs such as the liver. Herein, we show that dysfunction specifically in visceral adipose tissue was associated with fatty liver disease

Severe metabolic alterations in liver cancer lead to ERK pathway activation and drug resistance

Background: The extracellular signal-regulated kinase (ERK) pathway regulates cell growth, and is hyper-activated and associated with drug resistance in hepatocellular carcinoma (HCC). Metabolic pathways are profoundly dysregulated in HCC. Whether an altered metabolic state is linked to activated ERK pathway and drug response in HCC is unaddressed. Methods: We deprived HCC cells of glutamine to induce metabolic alterations and performed various assays, including metabolomics (with 13C-glucose isotope tracing), microarray analysis, and cell proliferation assays. Glutamine-deprived cells were also treated with kinase inhibitors (e.g. Sorafenib, Erlotinib, U0126 amongst other MEK inhibitors). We performed bioinformatics analysis and stratification of HCC tumour microarrays to determine upregulated ERK gene signatures in patients. Findings: In a subset of HCC cells, the withdrawal of glutamine triggers a severe metabolic alteration and ERK phosphorylation (pERK). This is accompanied by resistance to the anti-proliferative effect of kinase inhibitors, despite pERK inhibition. High intracellular serine is a consistent feature of an altered metabolic state and contributes to pERK induction and the kinase inhibitor resistance. Blocking the ERK pathway facilitates cell proliferation by reprogramming metabolism, notably enhancing aerobic glycolysis. We have identified 24 highly expressed ERK gene signatures that their combined expression strongly indicates a dysregulated metabolic gene network in human HCC tissues. Interpretation: A severely compromised metabolism lead to ERK pathway induction, and primes some HCC cells to pro-survival phenotypes upon ERK pathway blockade. Our findings offer novel insights for understanding, predicting and overcoming drug resistance in liver cancer patients

Dietary lipid droplet structure in postnatal life improves hepatic energy and lipid metabolism in a mouse model for postnatal programming

Early-life diets may have a long-lasting impact on metabolic health. This study tested the hypothesis that an early-life diet with large, phospholipid-coated lipid droplets (Concept) induces sustained improvements of hepatic mitochondrial function and metabolism. Young C57BL/6j mice were fed Concept or control (CTRL) diet from postnatal day 15 (PN15) to PN42, followed by western style (WSD) or standard rodent diet (AIN) until PN98. Measurements comprised body composition, insulin resistance (HOMA-IR), tricarboxylic acid (TCA) cycle- and β-oxidation-related hepatic oxidative capacity using high-resolution respirometry, mitochondrial dynamics, mediators of insulin resistance (diacylglycerols, DAG) or ceramides) in subcellular compartments as well as systemic oxidative stress. Concept feeding increased TCA cycle-related respiration by 33% and mitochondrial fusion protein-1 by 65% at PN42 (both p 0.05). At PN98, CTRL, but not Concept, mice developed hyperinsulinemia (CTRL/AIN 0.22 ± 0.44 vs. CTRL/WSD 1.49 ± 0.53 nmol/l, p 0.05 and Concept/AIN 0.20 ± 0.38 vs. Concept/WSD 1.00 ± 0.29 nmol/l, n.s.) and insulin resistance after WSD (CTRL/AIN 107 ± 23 vs. CTRL/WSD 738 ± 284, p 0.05 and Concept/AIN 109 ± 24 vs. Concept/WSD 524 ± 157, n.s.). WSD-induced liver weight was 18% lower in adult Concept-fed mice and β-oxidation-related respiration was 69% higher (p 0.05; Concept/WSD vs. Concept/AIN) along with lower plasma lipid peroxides (CTRL/AIN 4.85 ± 0.28 vs. CTRL/WSD 5.73 ± 0.47 µmol/l, p 0.05 and Concept/AIN 4.49 ± 0.31 vs. Concept/WSD 4.42 ± 0.33 µmol/l, n.s.) and were in part protected from WSD-induced increase in hepatic cytosolic DAG C16:0/C18:1. Early-life feeding of Concept partly protected from WSD-induced insulin resistance and systemic oxidative stress, potentially via changes in specific DAG and mitochondrial function, highlighting the role of early life diets on metabolic health later in life

Orphan nuclear receptor ERRγ is a key regulator of human fibrinogen gene expression

<div><p>Fibrinogen, 1 of 13 coagulation factors responsible for normal blood clotting, is synthesized by hepatocytes. Detailed roles of the orphan nuclear receptors regulating fibrinogen gene expression have not yet been fully elucidated. Here, we identified estrogen-related receptor gamma (ERRγ) as a novel transcriptional regulator of human fibrinogen gene expression. Overexpression of ERRγ specially increased fibrinogen expression in human hepatoma cell line. Cannabinoid receptor types 1(CB1R) agonist arachidonyl-2'-chloroethylamide (ACEA) up-regulated transcription of fibrinogen via induction of ERRγ, whereas knockdown of ERRγ attenuated fibrinogen expression. Deletion analyses of the fibrinogen γ (FGG) gene promoter and ChIP assays revealed binding sites of ERRγ on human fibrinogen γ gene promoter. Moreover, overexpression of ERRγ was sufficient to increase fibrinogen gene expression, whereas treatment with GSK5182, a selective inverse agonist of ERRγ led to its attenuation in cell culture. Finally, fibrinogen and ERRγ gene expression were elevated in liver tissue of obese patients suggesting a conservation of this mechanism. Overall, this study elucidates a molecular mechanism linking CB1R signaling, ERRγ expression and fibrinogen gene transcription. GSK5182 may have therapeutic potential to treat hyperfibrinogenemia.</p></div

Severe metabolic alterations in liver cancer lead to ERK pathway activation and drug resistance

Background: The extracellular signal-regulated kinase (ERK) pathway regulates cell growth, and is hyper-activated and associated with drug resistance in hepatocellular carcinoma (HCC). Metabolic pathways are profoundly dysregulated in HCC. Whether an altered metabolic state is linked to activated ERK pathway and drug response in HCC is unaddressed. Methods: We deprived HCC cells of glutamine to induce metabolic alterations and performed various assays, including metabolomics (with 13C-glucose isotope tracing), microarray analysis, and cell proliferation assays. Glutamine-deprived cells were also treated with kinase inhibitors (e.g. Sorafenib, Erlotinib, U0126 amongst other MEK inhibitors). We performed bioinformatics analysis and stratification of HCC tumour microarrays to determine upregulated ERK gene signatures in patients. Findings: In a subset of HCC cells, the withdrawal of glutamine triggers a severe metabolic alteration and ERK phosphorylation (pERK). This is accompanied by resistance to the anti-proliferative effect of kinase inhibitors, despite pERK inhibition. High intracellular serine is a consistent feature of an altered metabolic state and contributes to pERK induction and the kinase inhibitor resistance. Blocking the ERK pathway facilitates cell proliferation by reprogramming metabolism, notably enhancing aerobic glycolysis. We have identified 24 highly expressed ERK gene signatures that their combined expression strongly indicates a dysregulated metabolic gene network in human HCC tissues. Interpretation: A severely compromised metabolism lead to ERK pathway induction, and primes some HCC cells to pro-survival phenotypes upon ERK pathway blockade. Our findings offer novel insights for understanding, predicting and overcoming drug resistance in liver cancer patients

Knockdown of ERRγ attenuates ACEA-mediated induction of fibrinogen.

<p><b>(A)</b> ACEA-mediated induction of fibrinogen expression. Huh7 cells were treated with ACEA (10 μM) for the indicated time periods. Total RNAs were extracted for qPCR analyses. <b>(B-C)</b> Huh7 cells were treated with ACEA (10 μM) for 24 h. Total protein was extracted for western blotting (B). Cell culture media were collected to determine fibrinogen levels (C). * <i>p</i><0.05, ** <i>p</i><0.01. All data are representative of at least three independent experiments. Error bars show SEM. <b>(D)</b> Huh7 cells were treatment with ACEA in the continued presence or absence of AM251 for 24 h. qPCR were performed to measure mRNA levels. * <i>p</i><0.05. ** <i>p</i><0.01. <b>(E-G)</b> qPCR (E) and western blot (F) analysis showing mRNA and protein levels of ERRγ, FGA, FGB, and FGG in Huh7 cells. Huh7 cells were infected with Ad-Usi or Ad-shERRγ for 48 h, followed by treatment with ACEA (10 μM). Cell culture media were collected to determine fibrinogen levels (G).Western blot images were cropped with a black cropping line. All gels for Western blot analysis were run under the same experimental conditions. Full uncropped blots are available as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182141#pone.0182141.s003" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182141#pone.0182141.s004" target="_blank">S2</a> Figs.</p