Inhibitory Effect of Styrylpyrone Extract of Phellinus linteus on Hepatic Steatosis in HepG2 Cells.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is estimated to be approximately about 25.24% of the population worldwide. NAFLD is a complex syndrome and is characterized by a simple benign hepatocyte steatosis to more severe steatohepatitis in the liver pathology. Phellinus linteus (PL) is traditionally used as a hepatoprotective supplement. Styrylpyrone-enriched extract (SPEE) obtained from the PL mycelia has been shown to have potential inhibition effects on high-fat- and high-fructose-diet-induced NAFLD. In the continuous study, we aimed to explore the inhibitory effects of SPEE on free fatty acid mixture O/P [oleic acid (OA): palmitic acid (PA); 2:1, molar ratio]-induced lipid accumulation in HepG2 cells. Results showed that SPEE presented the highest free radical scavenging ability on DPPH and ABTS, and reducing power on ferric ions, better than that of partitions obtained from n-hexane, n-butanol and distilled water. In free-fatty-acid-induced lipid accumulation in HepG2 cells, SPEE showed an inhibition effect on O/P-induced lipid accumulation of 27% at a dosage of 500 µg/mL. As compared to the O/P induction group, the antioxidant activities of superoxide dismutase, glutathione peroxidase and catalase were enhanced by 73%, 67% and 35%, respectively, in the SPEE group. In addition, the inflammatory factors (TNF-α, IL-6 and IL-1ß) were significantly down-regulated by the SPEE treatment. The expressions of anti-adipogenic genes involved in hepatic lipid metabolism of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were enhanced in the SPEE supplemented HepG2 cells. In the protein expression study, p-AMPK, SIRT1 and PGC1-α were significantly increased to 121, 72 and 62%, respectively, after the treatment of SPEE. Conclusively, the styrylpyrone-enriched extract SPEE can ameliorate lipid accumulation and decrease inflammation and oxidative stress through the activation of SIRT1/AMPK/PGC1-α pathways.

Hyperuricemia induces lipid disturbances by upregulating the CXCL-13 pathway.

The molecular mechanism underlying hyperuricemia-induced lipid metabolism disorders is not clear. The purpose of the current study was to investigate the mechanism of lipid disturbances in a hyperuricemia mice model. RNA-Seq showed that differentially expressed genes (DEGs) in the fatty acid synthesis signaling pathway were mainly enriched and CXCL-13 was significantly enriched in protein-protein interaction networks. Western blotting, Q-PCR, and immunofluorescence results further showed that hyperuricemia upregulated CXCL-13 and disturbed lipid metabolism in vivo and in vitro. Furthermore, CXCL-13 alone also promoted the accumulation of lipid droplets and upregulated the expression of FAS and SREBP1, blocking AMPK signaling and activating the PKC and P38 signaling pathways. Silencing CXCL-13 reversed uric-acid-induced lipid droplet accumulation, which further downregulated FAS and SREBP1 expression, inhibited the p38 and PKC signaling, and activated AMPK signaling. In conclusion, hyperuricemia induces lipid metabolism disorders via the CXCL-13 pathway, making CXCL-13 a key regulatory factor linking hyperuricemia and lipid metabolism disorders. These results may provide novel insights for the treatment of hyperuricemia.NEW & NOTEWORTHY The underlying molecular mechanism of hyperuricemia-induced lipid metabolism disorders is still unclear. The study aimed to investigate the mechanism of lipid disturbance in hyperuricemia mice model. To our knowledge, we proposed for the first time that CXCL-13 may be a key regulator of hyperuricemia and lipid metabolism disorders. These results may provide new insights for the clinical treatment of hyperuricemia.

Liver X Receptor Alpha Activation Inhibits Autophagy and Lipophagy in Hepatocytes by Dysregulating Autophagy-Related 4B Cysteine Peptidase and Rab-8B, Reducing Mitochondrial Fuel Oxidation.

BACKGROUND AND AIMS: Fat accumulation results from increased fat absorption and/or defective fat metabolism. Currently, the lipid-sensing nuclear receptor that controls fat utilization in hepatocytes is elusive. Liver X receptor alpha (LXRα) promotes accumulation of lipids through the induction of several lipogenic genes. However, its effect on lipid degradation is open for study. Here, we investigated the inhibitory role of LXRα in autophagy/lipophagy in hepatocytes and the underlying basis. APPROACH AND RESULTS: In LXRα knockout mice fed a high-fat diet, or cell models, LXRα activation suppressed the function of mitochondria by inhibiting autophagy/lipophagy and induced hepatic steatosis. Gene sets associated with "autophagy" were enriched in hepatic transcriptome data. Autophagy flux was markedly augmented in the LXRα knockout mouse liver and primary hepatocytes. Mechanistically, LXRα suppressed autophagy-related 4B cysteine peptidase (ATG4B) and Rab-8B, responsible for autophagosome and -lysosome formation, by inducing let-7a and microRNA (miR)-34a. Chromatin immunoprecipitation assay enabled us to find LXRα as a transcription factor of let-7a and miR-34a. Moreover, 3' untranslated region luciferase assay substantiated the direct inhibitory effects of let-7a and miR-34a on ATG4B and Rab-8B. Consistently, either LXRα activation or the let-7a/miR-34a transfection lowered mitochondrial oxygen consumption rate and mitochondrial transmembrane potential and increased fat levels. In obese animals or nonalcoholic fatty liver disease (NAFLD) patients, let-7a and miR-34a levels were elevated with simultaneous decreases in ATG4B and Rab-8B levels. CONCLUSIONS: LXRα inhibits autophagy in hepatocytes through down-regulating ATG4B and Rab-8B by transcriptionally activating microRNA let-7a-2 and microRNA 34a genes and suppresses mitochondrial biogenesis and fuel consumption. This highlights a function of LXRα that culminates in the progression of liver steatosis and steatohepatitis, and the identified targets may be applied for a therapeutic strategy in the treatment of NAFLD.

Mallotucin D, a Clerodane Diterpenoid from Croton crassifolius, Suppresses HepG2 Cell Growth via Inducing Autophagic Cell Death and Pyroptosis.

Hepatocellular carcinoma (HCC) is a major subtype of primary liver cancer with a high mortality rate. Pyroptosis and autophagy are crucial processes in the pathophysiology of HCC. Searching for efficient drugs targeting pyroptosis and autophagy with lower toxicity is useful for HCC treatment. Mallotucin D (MLD), a clerodane diterpenoid from Croton crassifolius, has not been previously reported for its anticancer effects in HCC. This study aims to evaluate the inhibitory effects of MLD in HCC and explore the underlying mechanism. We found that the cell proliferation, DNA synthesis, and colony formation of HepG2 cells and the angiogenesis of HUVECs were all greatly inhibited by MLD. MLD caused mitochondrial damage and decreased the TOM20 expression and mitochondrial membrane potential, inducing ROS overproduction. Moreover, MLD promoted the cytochrome C from mitochondria into cytoplasm, leading to cleavage of caspase-9 and caspase-3 inducing GSDMD-related pyroptosis. In addition, we revealed that MLD activated mitophagy by inhibiting the PI3K/AKT/mTOR pathway. Using the ROS-scavenging reagent NAC, the activation effects of MLD on pyroptosis- and autophagy-related pathways were all inhibited. In the HepG2 xenograft model, MLD effectively inhibited tumor growth without detectable toxicities in normal tissue. In conclusion, MLD could be developed as a candidate drug for HCC treatment by inducing mitophagy and pyroptosis via promoting mitochondrial-related ROS production.

Arginine-Arginine-Leucine Peptide Targeting Heat Shock Protein 70 for Cancer Imaging.

Arg-Arg-Leu (RRL) is a potent tumor-homing tripeptide. However, the binding target is unclear. In this study, we intended to identify the binding target of RRL and evaluate the tumor targeting of 99mTc-MAG3-RRL in vivo. Biotin-RRL, 5-TAMRA-RRL, and 99mTc-MAG3-RRL were designed to trace the binding target and tumor lesion. Immunoprecipitation-mass spectrometry was conducted to identify the candidate proteins and determination of the subcellular localization was also performed. A pull-down assay was performed to demonstrate the immunoprecipitate. Fluorescence colocalization and cell uptake assays were performed to elucidate the correlation between the selected binding protein and RRL, and the internalization mechanism of RRL. Biodistribution and in vivo imaging were performed to evaluate the tumor accumulation and targeting of 99mTc-MAG3-RRL. The target for RRL was screened to be heat shock protein 70 (HSP70). The prominent uptake distribution of RRL was concentrated in the membrane and cytoplasm. A pull-down assay demonstrated the existence of HSP70 in the biotin-RRL captured complex. Regarding fluorescence colocalization and cell uptake assays, RRL may interact with HSP70 at the nucleotide-binding domain (NBD). Clathrin-dependent endocytosis and macropinocytosis could be a vital internalization mechanism of RRL. In vivo imaging and biodistribution both demonstrated that 99mTc-MAG3-RRL can trace tumors with satisfactory accumulation in hepatoma xenograft mice. The radioactive signals accumulated in tumor lesions can be blocked by VER-155008, which can bind to the NBD of HSP70. Our findings revealed that RRL may interact with HSP70 and that 99mTc-MAG3-RRL could be a prospective probe for visualizing overexpressed HSP70 tumor sections.

Apple polyphenol extract alleviates lipid accumulation in free-fatty-acid-exposed HepG2 cells via activating autophagy mediated by SIRT1/AMPK signaling.

Defective degradation of intracellular lipids induced by autophagy is causally linked to the development of non-alcoholic fatty liver disease (NAFLD). Natural agents that can restore autophagy could therefore have the potentials for clinical applications for this public health issue. Herein, we investigated the effects of apple polyphenol extract (APE) on fatty acid-induced lipids depositions in HepG2 cells. APE treatment alleviated palmitic acid and oleic acid-induced intracellular lipid accumulation, concomitant with the increased autophagy, restored lysosomal acidification, inhibited lipid synthesis and slight promotion of fatty acid oxidation. Mechanistically, APE up-regulated the expression of SIRT1, activated LKB1/AMPK pathway and inhibited mTOR signaling. Over-expressed or knock-down SIRT1 positively regulated AMPK and ATG7 expressions. SIRT1 and ATG7 knock-down impaired APE induction of improved lipid accumulation, increased intracellular TG content. Thus, APE induction of autophagy to ameliorate fatty acid-induced lipid deposition is SIRT1 dependent, APE conserved preventive potentials for clinical hepatosteatosis.

Dual Effect of Taxifolin on ZEB2 Cancer Signaling in HepG2 Cells.

Polyphenols, secondary metabolites of plants, exhibit different anti-cancer and cytoprotective properties such as anti-radical, anti-angiogenic, anti-inflammation, or cardioprotective. Some of these activities could be linked to modulation of miRNAs expression. MiRNAs play an important role in posttranscriptional regulation of their target genes that could be important within cell signalling or preservation of cell homeostasis, e.g., cell survival/apoptosis. We evaluated the influence of a non-toxic concentration of taxifolin and quercetin on the expression of majority human miRNAs via Affymetrix GeneChip™ miRNA 3.0 Array. For the evaluation we used two cell models corresponding to liver tissue, Hep G2 and primary human hepatocytes. The array analysis identified four miRNAs, miR-153, miR-204, miR-211, and miR-377-3p, with reduced expression after taxifolin treatment. All of these miRNAs are linked to modulation of ZEB2 expression in various models. Indeed, ZEB2 protein displayed upregulation after taxifolin treatment in a dose dependent manner. However, the modulation did not lead to epithelial mesenchymal transition. Our data show that taxifolin inhibits Akt phosphorylation, thereby diminishing ZEB2 signalling that could trigger carcinogenesis. We conclude that biological activity of taxifolin may have ambiguous or even contradictory outcomes because of non-specific effect on the cell.

Glial Cell Line-Derived Neurotrophic Factor Enhances Autophagic Flux in Mouse and Rat Hepatocytes and Protects Against Palmitate Lipotoxicity.

Glial cell line-derived neurotrophic factor (GDNF) is a protein that is required for the development and survival of enteric, sympathetic, and catecholaminergic neurons. We previously reported that GDNF is protective against high fat diet (HFD)-induced hepatic steatosis in mice through suppression of hepatic expression of peroxisome proliferator activated receptor-γ and genes encoding enzymes involved in de novo lipogenesis. We also reported that transgenic overexpression of GDNF in mice prevented the HFD-induced liver accumulation of the autophagy cargo-associated protein p62/sequestosome 1 characteristic of impaired autophagy. Here we investigated the effects of GDNF on hepatic autophagy in response to increased fat load, and on hepatocyte mitochondrial fatty acid ß-oxidation and cell survival. GDNF not only prevented the reductions in the liver levels of some key autophagy-related proteins, including Atg5, Atg7, Beclin-1 and LC3A/B-II, seen in HFD-fed control mice, but enhanced their levels after 12 weeks of HFD feeding. In vitro, GDNF accelerated autophagic cargo clearance in primary mouse hepatocytes and a rat hepatocyte cell line, and reduced the phosphorylation of the mechanistic target of rapamycin complex downstream-target p70S6 kinase similar to the autophagy activator rapamycin. GDNF also enhanced mitochondrial fatty acid ß-oxidation in primary mouse and rat hepatocytes, and protected against palmitate-induced lipotoxicity. Conclusion: We demonstrate a role for GDNF in enhancing hepatic autophagy and in potentiating mitochondrial function and fatty acid oxidation. Our studies show that GDNF and its receptor agonists could be useful for enhancing hepatocyte survival and protecting against fatty acid-induced hepatic lipotoxicity.

Reactive oxygen species generation and mitochondrial dysfunction for the initiation of apoptotic cell death in human hepatocellular carcinoma HepG2 cells by a cyclic dipeptide Cyclo(-Pro-Tyr).

Cyclic dipeptides are increasingly gaining importance as considering its significant biological and pharmacological activities. This study was aimed to investigate the anticancer activity of a dipeptide Cyclo(-Pro-Tyr) (DP) identified from marine sponge Callyspongia fistularis symbiont Bacillus pumilus AMK1 and the underlying apoptotic mechanisms in the liver cancer HepG2 cell lines. MTT assay was done to demonstrate the cytotoxic effect of DP in HepG2 cells and mouse Fibroblast McCoy cells. Initially, apoptosis inducing activity of DP was identified using propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) dual staining, then it was confirmed by DNA fragmentation assay and western blotting analysis of apoptosis related markers Bax, Bcl-2, cytochrome c, caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Rhodamine 123 staining was performed to observe DP effects on the mitochondrial membrane potential (MMP) and DCFH-DA (Dichloro-dihydro-fluorescein diacetate) staining was done to measure the intracellular reactive oxygen species (ROS) levels. The MTT results revealed that DP initiated dose-dependent cytotoxicity in HepG2 cells, but no significant toxicity in mouse Fibroblast McCoy cells treated with DP at the specified concentrations. DP induced apoptosis, which is confirmed by the appearance of apoptotic bodies with PI and AO/EB dual staining, and DNA fragmentation. DP significantly elevated the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), enhanced cytochrome c release from mitochondria, increased caspase-3 activation, the cleavage of PARP and increased intracellular reactive oxygen species (ROS) levels. Besides this, DP successfully inhibited the phosphorylation of PI3K, AKT and increased PTEN expression. These results suggested DP might have anti-cancer effect by initiating apoptosis through mitochondrial dysfunction and downregulating PI3K/Akt signaling pathway in HepG2 cells with no toxicity effect on normal fibroblast cells. Therefore, DP may be developed as a potential alternative therapeutic agent for treating hepatocellular carcinoma.

Primary Human Hepatocytes, but Not HepG2 or Balb/c 3T3 Cells, Efficiently Metabolize Salinomycin and Are Resistant to Its Cytotoxicity.

Salinomycin is a polyether antibiotic showing anticancer activity. There are many reports of its toxicity to animals but little is known about the potential adverse effects in humans. The action of the drug may be connected to its metabolism. That is why we investigated the cytotoxicity of salinomycin and pathways of its biotransformation using human primary hepatocytes, human hepatoma cells (HepG2), and the mouse fibroblast cell line (Balb/c 3T3). The cytotoxicity of salinomycin was time-dependent, concentration-dependent, and cell-dependent with primary hepatocytes being the most resistant. Among the studied models, primary hepatocytes were the only ones to efficiently metabolize salinomycin but even they were saturated at higher concentrations. The main route of biotransformation was monooxygenation leading to the formation of monohydroxysalinomycin, dihydroxysalinomycin, and trihydroxysalinomycin. Tiamulin, which is a known inhibitor of CYP450 izoenzymes, synergistically induced cytotoxicity of salinomycin in all cell types, including non-metabolising fibroblasts. Therefore, the pharmacokinetic interaction cannot fully explain tiamulin impact on salinomycin toxicity.

The distal upstream region of insulin-like growth factor-binding protein-1 enhances its expression in endometrial stromal cells during decidualization.

We have previously shown that decidualization of human endometrial stromal cells (ESCs) causes a genome-wide increase in the levels of acetylation of histone-H3 Lys-27 (H3K27ac). We also reported that the distal gene regions, more than 3 kb up- or downstream of gene transcription start sites have increased H3K27ac levels. Insulin-like growth factor-binding protein-1 (IGFBP-1) is a specific decidualization marker and has increased H3K27ac levels in its distal upstream region (-4701 to -7501 bp). Here, using a luciferase reporter gene construct containing this IGFBP-1 upstream region, we tested the hypothesis that it is an IGFBP-1 enhancer. To induce decidualization, we incubated ESCs with cAMP and found that cAMP increased luciferase expression, indicating that decidualization increased the transcriptional activity from the IGFBP-1 upstream region. Furthermore, CRISPR/Cas9-mediated deletion of this region in HepG2 cells significantly reduced IGFBP-1 expression, confirming its role as an IGFBP-1 enhancer. A ChIP assay revealed that cAMP increased the recruitment of the transcriptional regulators CCAAT enhancer-binding protein ß (C/EBPß), forkhead box O1 (FOXO1), and p300 to the IGFBP-1 enhancer in ESCs. Of note, C/EBPß knockdown inhibited the stimulatory effects of cAMP on the levels of H3K27ac, chromatin opening, and p300 recruitment at the IGFBP-1 enhancer. These results indicate that the region -4701 to -7501 bp upstream of IGFBP-1 functions as an enhancer for IGFBP-1 expression in ESCs undergoing decidualization, that C/EBPß and FOXO1 bind to the enhancer region to up-regulate IGFBP-1 expression, and that C/EBPß induces H3K27ac by recruiting p300 to the IGFBP-1 enhancer.

LINC01133 aggravates the progression of hepatocellular carcinoma by activating the PI3K/AKT pathway.

LncRNAs exhibit crucial roles in various pathological diseases, including hepatocellular carcinoma (HCC). Therefore, it is significant to recognize the dysregulated lncRNAs in HCC progression. Recently, LINC01133 has been identified in several tumors. However, the biological role of LINC01133 in HCC remains poorly understood. Currently, we focused on the function of LINC01133 in HCC development. We observed that LINC01133 was significantly increased in HCC cells including HepG2, Hep3B, MHCC-97L, SK-Hep-1, and MHCC-97H cells compared with the normal human liver cell line HL-7702. In addition, PI3K/AKT signaling was highly activated in HCC cells. Knockdown of LINC01133 was able to inhibit HCC cell proliferation, cell colony formation, cell apoptosis, and blocked cell cycle arrest in the G1 phase. For another, downregulation of LINC01133 repressed HCC cell migration and invasion. Subsequently, the PI3K/AKT signaling pathway was strongly suppressed by silence of LINC01133 in Hep3B and HepG2 cells. Then, in vivo tumor xenografts models were established using Hep3B cells to explore the function of LINC01133 in HCC progression. Consistently, our study indicated that knockdown of LINC01133 dramatically repressed HCC tumor progression through targeting the PI3K/AKT pathway in vivo. Taken these together, we revealed that LINC01133 contributed to HCC progression by activating the PI3K/AKT pathway.

Cell identity bookmarking through heterogeneous chromatin landscape maintenance during the cell cycle.

Genetic and epigenetic information are faithfully duplicated and accurately transmitted to daughter cells to preserve cell identity during the cell cycle. However, how the chromatin-based epigenetic information beyond DNA sequence is stably transmitted along with the disruption and re-establishment of chromatin structure within a cell cycle remains largely unexplored. Through comprehensive analysis DNA methylation and nucleosome positioning patterns of HepG2 cells in G0/G1, early S, late S and G2/M phases, we found that DNA methylation may act as the prime element for epigenetic inheritance after replication, as DNA methylation was extremely stable in each cell cycle phase, while nucleosome occupancy showed notable phase dependent fluctuation. Nucleosome-Secured Regions (NSRs) occupied by polycomb-repressed chromatin played a role in repressing the irrelevant cell type-specific genes and were essential for preventing irrelevant transcription factors binding, while the well-defined Nucleosome-Depleted Regions (NDRs) marked the genes crucial for cell identity maintenance. Chromatin structure at NSRs and NDRs was well maintained throughout the cell cycle, which played crucial roles in steadily preserving the transcriptional identity of the cell to fulfill cell identity maintenance. Collectively, our results demonstrated that while chromatin architecture underwent dynamic changes during cell cycle progression, DNA methylation together with NSRs and NDRs were stable epigenetic elements that were required for faithful transmission to the daughter cell to accurately maintain cell identity during the cell cycle.

The FUT2 secretor variant p.Trp154Ter influences serum vitamin B12 concentration via holo-haptocorrin, but not holo-transcobalamin, and is associated with haptocorrin glycosylation.

Vitamin B12 deficiency is common in older individuals. Circulating vitamin B12 concentration can be used to diagnose deficiency, but this test has substantial false positive and false negative rates. We conducted genome-wide association studies (GWAS) in which we resolved total serum vitamin B12 into the fractions bound to transcobalamin and haptocorrin: two carrier proteins with very different biological properties. We replicated reported associations between total circulating vitamin B12 concentrations and a common null variant in FUT2. This allele determines the secretor phenotype in which blood group antigens are found in non-blood body fluids. Vitamin B12 bound to haptocorrin (holoHC) remained highly associated with FUT2 rs601338 (p.Trp154Ter). Transcobalamin bound vitamin B12 (holoTC) was not influenced by this variant. HoloTC is the bioactive the form of the vitamin and is taken up by all tissues. In contrast, holoHC is only taken up by the liver. Using holoHC from individuals with known FUT2 genotypes, we demonstrated that FUT2 rs601338 genotype influences the glycosylation of haptocorrin. We then developed an experimental model demonstrating that holoHC is transported into cultured hepatic cells (HepG2) via the asialoglycoprotein receptor (ASGR). Our data challenge current published hypotheses on the influence of genetic variation on this clinically important measure and are consistent with a model in which FUT2 rs601338 influences holoHC by altering haptocorrin glycosylation, whereas B12 bound to non-glycosylated transcobalamin (i.e. holoTC) is not affected. Our findings explain some of the observed disparity between use of total B12 or holoTC as first-line clinical tests of vitamin B12 status.

ROS -mediated p53 activation by juglone enhances apoptosis and autophagy in vivo and in vitro.

Juglone (JG) exhibits a broad-spectrum of cytotoxicity against some cancer cells. However, its molecular mechanisms have not been investigated well. Here, the present results showed that JG significantly inhibited tumor growth in vivo. CCK-8 assays, flow cytometric analysis, western blotting and immunohistochemistry revealed that JG effectively inhibited cell proliferation and induced apoptosis through extrinsic pathways. We also observed that JG treatment induced autophagy flux via activiting the AMPK-mTOR signaling pathway. In addition, we found that JG enhanced p53 activation by increasing down-regulation of ubiquitin-mediated degradation. Inhibition of p53 by siRNA attenuated JG-induced cell death and autophagy. Moreover, JG enhanced the generation of hydrogen peroxide (H2O2) and superoxide anion radical (O2• -). Further experiments proved that H2O2 was a major factor since the H2O2 scavenger catalase (CAT) reduced both autophagy and cell death to a greater extent than the O2• - scavenger SOD. Overall, our results illustrated that JG caused apoptosis and autophagy via activating the ROS-mediated p53 pathway in human liver cancer cells in vitro and in vivo, which provided basic scientific evidence that JG serves as a potential anti-cancer agent.

Influence of total polar compounds on lipid metabolism, oxidative stress and cytotoxicity in HepG2 cells.

BACKGROUND: Recently, the harmful effects of frying oil on health have been gradually realized. However, as main components of frying oils, biochemical effects of total polar compounds (TPC) on a cellular level were underestimated. METHODS: The effects of total polar compounds (TPC) in the frying oil on the lipid metabolism, oxidative stress and cytotoxicity of HepG2 cells were investigated through a series of biochemical methods, such as oil red staining, real-time polymerase chain reaction (RT-PCR), cell apoptosis and cell arrest. RESULTS: Herein, we found that the survival rate of HepG2 cells treated with TPC decreased in a time and dose dependent manner, and thereby presented significant lipid deposition over the concentration of 0.5 mg/mL. TPC were also found to suppress the expression levels of PPARα, CPT1 and ACOX, elevate the expression level of MTP and cause the disorder of lipid metabolism. TPC ranged from 0 to 2 mg/mL could significantly elevate the amounts of reactive oxygen species (ROS) in HepG2 cells, and simultaneously increase the malondialdehyde (MDA) content from 21.21 ± 2.62 to 65.71 ± 4.20 µmol/mg of protein (p < 0.05) at 24 h. On the contrary, antioxidant enzymes superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) respectively decreased by 0.52-, 0.56- and 0.28-fold, when HepG2 cells were exposed to 2 mg/mL TPC for 24 h. In addition, TPC could at least partially induce the apoptosis of HepG2 cells, and the transition from G0/G1 to G2 phase in HepG2 cells was impeded. CONCLUSIONS: TPC could progressively cause lipid deposition, oxidative stress and cytotoxicity, providing the theoretical support for the detrimental health effects of TPC.

PPARα and γ Activation Effects of New Nor-triterpenoidal Saponins from the Aerial Parts of Anabasis articulata.

Anabasis articulata, traditionally used to treat diabetes, is rich in saponin content. This study was performed to investigate the agonistic effect of its saponins on peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor-γ in human hepatoma (HepG2) cells to explore the possibility of the involvement of these nuclear receptors in the mechanism of the antidiabetic effect of the plant. Chemical investigation of the n-butanol fraction resulted in the isolation of three new and one known 30-noroleanane triterpenoid saponins. The structures of the new compounds were elucidated as 3ß-hydroxy,23-aldehyde-30-norolean-12,20(29)-dien-28-oic acid-28-O-ß-D-glucopyranosyl ester (1: ), 3ß-O-D-galactopyranosyl-23-aldehyde-30-norolean-12,20(29)-dien-28-oic acid-28-O-ß-D-glucopyranosyl ester (2: ), and 3ß-O-D-xylopyranosyl-30-norolean-12,20(29)-dien-28-oic acid 28-O-ß-D-glucopyranosyl ester (3: ), while the known 30-nortriterpenoidal saponin was identified as boussingoside E (4: ). Although, the isolated saponins (1:  - 4: ) did not show > 1.5-fold activation of peroxisome proliferator-activated receptor-γ, but two of them (1: and 3: ) activated peroxisome proliferator-activated receptor-α to the higher extents of 2.25- and 1.86-fold, respectively. These results suggest that the reported antidiabetic action of the isolated saponins may not solely involve the activation of peroxisome proliferator-activated receptor-γ. However, the agonistic activity of the n-butanol fraction of A. articulata (1.71-fold induction) and two of its saponins (1: and 3: ) towards peroxisome proliferator-activated receptor-α may be beneficial in the cardiovascular condition that is closely associated with diabetes and other metabolic disorders.

Acetyl-CoA carboxylase inhibition by ND-630 reduces hepatic steatosis, improves insulin sensitivity, and modulates dyslipidemia in rats.

Simultaneous inhibition of the acetyl-CoA carboxylase (ACC) isozymes ACC1 and ACC2 results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation and may favorably affect the morbidity and mortality associated with obesity, diabetes, and fatty liver disease. Using structure-based drug design, we have identified a series of potent allosteric protein-protein interaction inhibitors, exemplified by ND-630, that interact within the ACC phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit the enzymatic activity of both ACC isozymes, reduce fatty acid synthesis and stimulate fatty acid oxidation in cultured cells and in animals, and exhibit favorable drug-like properties. When administered chronically to rats with diet-induced obesity, ND-630 reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia. When administered chronically to Zucker diabetic fatty rats, ND-630 reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c (0.9% reduction). Together, these data suggest that ACC inhibition by representatives of this series may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes mellitus, and fatty liver disease.

PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The covalently closed circular DNA (cccDNA) minichromosome, which serves as the template for the transcription of viral RNAs, plays a key role in viral persistence. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation of cccDNA-bound histone 3 (H3) and H4, the potential contributions of histone methylation and related host factors remain obscure. Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as an effective restrictor of HBV transcription and replication. In cell culture-based models for HBV infection and in liver tissues of patients with chronic HBV infection, we found that symmetric dimethylation of arginine 3 on H4 on cccDNA was a repressive marker of cccDNA transcription and was regulated by PRMT5 depending on its methyltransferase domain. Moreover, PRMT5-triggered symmetric dimethylation of arginine 3 on H4 on the cccDNA minichromosome involved an interaction with the HBV core protein and the Brg1-based human SWI/SNF chromatin remodeler, which resulted in down-regulation of the binding of RNA polymerase II to cccDNA. In addition to the inhibitory effect on cccDNA transcription, PRMT5 inhibited HBV core particle DNA production independently of its methyltransferase activity. Further study revealed that PRMT5 interfered with pregenomic RNA encapsidation by preventing its interaction with viral polymerase protein through binding to the reverse transcriptase-ribonuclease H region of polymerase, which is crucial for the polymerase-pregenomic RNA interaction. CONCLUSION: PRMT5 restricts HBV replication through a two-part mechanism including epigenetic suppression of cccDNA transcription and interference with pregenomic RNA encapsidation; these findings improve the understanding of epigenetic regulation of HBV transcription and host-HBV interaction, thus providing new insights into targeted therapeutic intervention. (Hepatology 2017;66:398-415).

Functional lipidomics: Palmitic acid impairs hepatocellular carcinoma development by modulating membrane fluidity and glucose metabolism.

Lipids are essential cellular components and energy sources of living organisms, and altered lipid composition is increasingly recognized as a signature of cancer. We performed lipidomic analysis in a series of hepatocellular carcinoma (HCC) cells and identified over 1,700 intact lipids originating from three major lipid categories. Comparative lipidomic screening revealed that 93 significantly changed lipids and decreased palmitic acyl (C16:0)-containing glycerophospholipids were positively associated with metastatic abilities of HCC cells. Furthermore, both in vitro and in vivo experiments demonstrated that C16:0 incubation specifically reduced malignant cell proliferation, impaired cell invasiveness, and suppressed tumor growth in mouse xenograft models. Biochemical experiments demonstrated that C16:0 treatment decreased cell membrane fluidity and limited glucose metabolism. A phosphoproteomics approach further revealed such C16:0 incubation attenuated phosphorylation levels of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) pathway proteins. Multiple reaction monitoring analysis of 443 lipid molecules showed 8 reduced C16:0-containing lipids out of total 10 altered lipids when cancer tissues were compared with adjacent nontumor tissues in a cohort of clinical HCC specimens (P < 0.05). CONCLUSION: These data collectively demonstrate the biomedical potential of using altered lipid metabolism as a diagnostic marker for cancerous cells and open an opportunity for treating aggressive HCCs by targeting altered C16:0 metabolism. (Hepatology 2017;66:432-448).