Human Hepatoma-derived Cell Research Articles

Impact of miR-222-3p-mediated downregulation of arylacetamide deacetylase on drug hydrolysis and lipid accumulation

Arylacetamide deacetylase (AADAC) is involved in drug hydrolysis and lipid metabolism. In 23 human liver samples, no significant correlation was observed between AADAC mRNA (19.7-fold variation) and protein levels (137.6-fold variation), suggesting a significant contribution of post-transcriptional regulation to AADAC expression. The present study investigated whether AADAC is regulated by microRNA in the human liver and elucidate its biological significance. Computational analysis predicted two potential miR-222-3p recognition elements in the 3′-untranslated region (UTR) of AADAC. Luciferase assay revealed that the miR-222-3p recognition element was functional in downregulating AADAC expression. In HEK293 cells transfected with an AADAC expression plasmid containing 3′-UTR, miR-222-3p overexpression decreased AADAC protein level and activity, whereas miR-222-3p inhibition increased them. Similar results were observed in human hepatoma-derived Huh-1 cells endogenously expressing AADAC and HepaSH cells that are hepatocytes from chimeric mice with humanized livers. In individual human liver samples, AADAC protein levels inversely correlated with miR-222-3p levels. Overexpression of miR-222-3p resulted in increased lipid accumulation in Huh-1 cells, which was reversed by AADAC overexpression. In contrast, miR-222-3p inhibition decreased lipid accumulation, which was reversed by AADAC knockdown. In conclusion, we found that hepatic AADAC was downregulated by miR-222-3p, resulting in decreased drug hydrolysis and increased lipid accumulation.

Suppression of Hyperglycemia-Induced Insulin Resistance by Rapanone Isolated from Connarus Ruber

Although it would be ideal if insulin resistance due to a persistent hyperglycemic state could be prevented by consuming particular foods, to the best of our knowledge, no foods with such effects have as yet been reported. Connarus ruber (CR) extract, previously indicated to be effective against diabetes, was investigated in this study to ascertain whether it can inhibit the induction of insulin resistance in human hepatoma-derived HepG2 cells. Insulin resistance can be induced in cells by cultivating under hyperglycemic conditions. HepG2 cells were cultured for 24 and 48 h with 1.25 µg/mL of CR extract and its components, rapanone and embelin, at 1.25 and 5 µg/mL, followed by the induction of insulin resistance by hyperglycemic conditions. The results revealed a decrease over time in the glucose concentration of HepG2 cells, indicating that insulin resistance was inhibited in these cells. However, when HepG2 cells, already insulin resistant owing to previous exposure to hyperglycemic conditions, were cultured for 24 and 48 h with CR extract containing rapanone and embelin at <10 µg/mL, the glucose concentration creased over time, indicating that insulin resistance was not affected under these conditions. The results suggest that insulin resistance is inhibited only when CR extract exposure occurs before exposure to hyperglycemia conditions and CR cannot induce recovery from insulin resistance. Thus, this study indicates that CR extract, with rapanone and embelin as active ingredients, could be used as a functional food to reduce the risk of developing type 2 diabetes mellitus as a result of chronic hyperglycemia.

Characterization of CCoV-HuPn-2018 spike protein-mediated viral entry.

Canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) was recently isolated from a child with pneumonia. This novel human pathogen resulted from cross-species transmission of a canine coronavirus. It has been known that CCoV-HuPn-2018 uses aminopeptidase N (APN) from canines, felines, and porcines, but not humans, as functional receptors for cell entry. The molecular mechanism of cell entry in CCoV-HuPn-2018 remains poorly understood. In this study, we demonstrated that among the nine APN orthologs tested, the APN of the Mexican free-tailed bat could also efficiently support CCoV-HuPn-2018 spike (S) protein-mediated entry, raising the possibility that bats may also be an alternative host epidemiologically important for the transmission of this virus. The glycosylation at residue N747 of canine APN is critical for its receptor activity. The gain of glycosylation at the corresponding residues in human and rabbit APNs converted them to functional receptors for CCoV-HuPn-2018. Interestingly, the CCoV-HuPn-2018 spike protein pseudotyped virus infected multiple human cancer cell lines in a human APN-independent manner, whereas sialic acid appeared to facilitate the entry of the pseudotyped virus into human cancer cells. Moreover, while host cell surface proteases trypsin and TMPRSS2 did not promote the entry of CCoV-HuPn-2018, endosomal proteases cathepsin L and B are required for the entry of CCoV-HuPn-2018 in a pH-dependent manner. IFITMs and LY6E are host restriction factors for the CCoV-HuPn-2018 entry. Our results thus suggest that CCoV-HuPn-2018 has not yet evolved to be an efficient human pathogen. Collectively, this study helps us understand the cell tropism, receptor usage, cross-species transmission, natural reservoir, and pathogenesis of this potential human coronavirus. IMPORTANCE Viral entry is driven by the interaction between the viral spike protein and its specific cellular receptor, which determines cell tropism and host range and is the major constraint to interspecies transmission of coronaviruses. Aminopeptidase N (APN; also called CD13) is a cellular receptor for HCoV-229E, the newly discovered canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018), and many other animal alphacoronaviruses. We examined the receptor activity of nine APN orthologs and found that CCoV-HuPn-2018 utilizes APN from a broad range of animal species, including bats but not humans, to enter host cells. To our surprise, we found that CCoV-HuPn-2018 spike protein pseudotyped viral particles successfully infected multiple human hepatoma-derived cell lines and a lung cancer cell line, which is independent of the expression of human APN. Our findings thus provide mechanistic insight into the natural hosts and interspecies transmission of CCoV-HuPn-2018-like coronaviruses.

Pharmacological activities of Artemisia absinthium and control of hepatic cancer by expression regulation of TGFβ1 and MYC genes.

Plant derived compounds have always been an important source of medicines and have received significant attention in recent years due to their diverse pharmacological properties. Millions of plant-based herbal or traditional medicines are used to cure various types of cancers especially due to activation of proliferative genes. The aim of the present study was to characterize the altered and attenuated gene expression of the selected growth factor namely Transforming growth factor Beta -1 (TGFβ1) and MYC in human hepatoma-derived (Huh7) liver cancer cell lines in response to extracts of Artemisia absinthium dissolved in selected organic solvents. Ethanolic, methanolic and acetone extract of different plant parts (leaf, stem and flowers) was used to access the antiproliferative activity by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Quantitative Real-Time RT-PCR revealed that the transcript levels of TGFβ1 are induced in the samples treated with methanolic extract of Artemisia absinthium. Furthermore, reduced expression levels of MYC gene was noticed in cancerous cells suggesting antiproliferative properties of the plant. This study further highlights the resistance profile of various microbes by antimicrobial susceptibility test with plant extracts. In addition, antidiabetic effect of Artemisia absinthium have also shown positive results. Our study elucidates the potentials of Artemisia absinthium as a medicinal plant, and highlights the differential expression of genes involved in its mitogenic and anti-proliferative activity with a brief account of its pharmacological action.

Silencing of STE20-type kinase TAOK1 confers protection against hepatocellular lipotoxicity through metabolic rewiring.

NAFLD has become the leading cause of chronic liver disease worldwide afflicting about one quarter of the adult population. NASH is a severe subtype of NAFLD, which in addition to hepatic steatosis connotes liver inflammation and hepatocyte ballooning. In light of the exponentially increasing prevalence of NAFLD, it is imperative to gain a better understanding of its molecular pathogenesis. The aim of this study was to examine the potential role of STE20-type kinase TAOK1 -a hepatocellular lipid droplet-associated protein-in the regulation of liver lipotoxicity and NAFLD etiology. The correlation between TAOK1 mRNA expression in liver biopsies and the severity of NAFLD was evaluated in a cohort of 62 participants. Immunofluorescence microscopy was applied to describe the subcellular localization of TAOK1 in human and mouse hepatocytes. Metabolic reprogramming and oxidative/endoplasmic reticulum stress were investigated in immortalized human hepatocytes, where TAOK1 was overexpressed or silenced by small interfering RNA, using functional assays, immunofluorescence microscopy, and colorimetric analysis. Migration, invasion, and epithelial-mesenchymal transition were examined in TAOK1-deficient human hepatoma-derived cells. Alterations in hepatocellular metabolic and pro-oncogenic signaling pathways were assessed by immunoblotting. We observed a positive correlation between the TAOK1 mRNA abundance in human liver biopsies and key hallmarks of NAFLD (i.e., hepatic steatosis, inflammation, and ballooning). Furthermore, we found that TAOK1 protein fully colocalized with intracellular lipid droplets in human and mouse hepatocytes. The silencing of TAOK1 alleviated lipotoxicity in cultured human hepatocytes by accelerating lipid catabolism (mitochondrial β-oxidation and triacylglycerol secretion), suppressing lipid anabolism (fatty acid influx and lipogenesis), and mitigating oxidative/endoplasmic reticulum stress, and the opposite changes were detected in TAOK1-overexpressing cells. We also found decreased proliferative, migratory, and invasive capacity, as well as lower epithelial-mesenchymal transition in TAOK1-deficient human hepatoma-derived cells. Mechanistic studies revealed that TAOK1 knockdown inhibited ERK and JNK activation and repressed acetyl-CoA carboxylase (ACC) protein abundance in human hepatocytes. Together, we provide the first experimental evidence supporting the role of hepatic lipid droplet-decorating kinase TAOK1 in NAFLD development through mediating fatty acid partitioning between anabolic and catabolic pathways, regulating oxidative/endoplasmic reticulum stress, and modulating metabolic and pro-oncogenic signaling.

CCT2 Gene Expression in Hepatocellular Carcinoma and its Effect on the Biological Function of Hepatocellular Carcinoma Cells

The main objective of the study is to investigate the expression level of chaperonin containing t-complex polypeptide 1 subunit 2 in hepatocellular carcinoma tissues and cell lines, and its effect on the proliferation, invasion and migration of hepatocellular carcinoma cells. Immunohistochemistry was used to detect the protein expression levels of chaperonin containing t-complex polypeptide 1 subunit 2 in 89 hepatocellular carcinoma tissues and tumor-adjacent tissues. Cell migration and invasion were detected by transwell assay. A subcutaneous xenograft model was constructed in nude mice to investigate the effect of chaperonin containing t-complex polypeptide 1 subunit 2 gene knockdown on tumor growth. Immunohistochemistry results indicated that 79.78 % of hepatocellular carcinoma patients had highly expressed chaperonin containing t-complex polypeptide 1 subunit 2 proteins and the expression in hepatocellular carcinoma tissues was significantly higher than that in tumor-adjacent tissues. The proliferation, invasion and migration of hepatoblastoma cell line HepG2 cells with chaperonin containing t-complex polypeptide 1 subunit 2 gene knockdown were inhibited, while those of human hepatoma-derived Huh-7 cells with chaperonin containing t-complex polypeptide 1 subunit 2 gene overexpression were enhanced. In vivo experiments also confirmed that the tumor growth rate in the chaperonin containing t-complex polypeptide 1 subunit 2 knockdown group was significantly slower than that in the vector group. Chaperonin containing t-complex polypeptide 1 subunit 2 is an independent poor prognostic factor of hepatocellular carcinoma and its expression is significantly upregulated in hepatocellular carcinoma tissues and cell lines. Chaperonin containing t-complex polypeptide 1 subunit 2 can significantly promote the proliferation, invasion and migration of hepatocellular carcinoma cells, suggesting that it can be an important therapeutic target for hepatocellular carcinoma.

Exogenous miRNAs from Moringa oleifera Lam. recover a dysregulated lipid metabolism.

A balanced diet is critical for human health, and edible plants play an important role in providing essential micronutrients as well as specific microRNAs (miRNAs) that can regulate human gene expression. Here we present the effects of Moringa oleifera (MO) miRNAs (mol-miRs) on lipid metabolism. Through in silico studies we identified the potential genes involved in lipid metabolism targeted by mol-miRs. To this end, we tested the efficacy of an aqueous extract of MO seeds (MOES), as suggested in traditional African ethnomedicine, or its purified miRNAs. The biological properties of MO preparations were investigated using a human derived hepatoma cell line (HepG2) as a model. MOES treatment decreased intracellular lipid accumulation and induced apoptosis in HepG2. In the same cell line, transfection with mol-miRs showed similar effects to MOES. Moreover, the effect of the mol-miR pool was investigated in a pre-obese mouse model, in which treatment with mol-miRs was able to prevent dysregulation of lipid metabolism.

천마 추출물을 처리한 간암 세포에서 Bcl-2, Bax 및 AMPKα 발현과세포 자멸사에 미치는 영향 분석

Background: This study was carried out to investigate the anticancer effects of Gastrodia elata Blume extract (GEE) in human hepatoma-derived Hep3B cells by apoptotic cell death.BRMethods and Results: To evaluate the anticancer effect, a Hep3B cell viability assay and a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) on GEE were performed, which was obtained extracted by ultrasound-assisted extraction and then used to treat the Hep3B cells. Cell viability was inhibited to 48.2% (4.0 ㎎/㎖). To identify anticancer mechanisms using qRT-PCR, mRNA expressions of B-cell lymphoma-2, its associated Bax protein, and 5’-AMP-activated protein kinase were examined with a comparative △CSUBT/SUB method. A qualitative analysis of the bioactive compounds in GEE was coducted using liquid chromatography tandem mass spectrometry, gastrodin and phloretin were identified as the two major compounds.BRConclusions: These findings demonstrate that GEE can decrease liver cancer cell viability and control apoptosis gene expression to have anticancer effects, GEE could be employed as a natural anticancer agent with less side effects than those of traditional anticancer medications.

Deciphering the molecular mechanisms underlying the increase in PCSK9 plasma levels in response to anti-PCSK9 mabs: In vitro study in human hepatoma-derived Huh7 cells

Background and Aims : In response to anti-PCSK9 monoclonal antibodies (mAbs) the circulating levels of total PCSK9 raised by approximately 4/7-fold (Shapiro et al. 2018). We aim to study the molecular mechanisms underlying this phenomenon.

Preclinical Efficacy and Toxicity Analysis of the Pan-Histone Deacetylase Inhibitor Gossypol for the Therapy of Colorectal Cancer or Hepatocellular Carcinoma.

Gossypol, a sesquiterpenoid found in cotton seeds, exerts anticancer effects on several tumor entities due to inhibition of DNA synthesis and other mechanisms. In clinical oncology, histone deacetylase inhibitors (HDACi) are applied as anticancer compounds. In this study, we examined whether gossypol harbors HDAC inhibiting activity. In vitro analyses showed that gossypol inhibited class I, II, and IV HDAC, displaying the capability to laterally interact with the respective catalytic center and is, therefore, classified as a pan-HDAC inhibitor. Next, we studied the effects of gossypol on human-derived hepatoma (HepG2) and colon carcinoma (HCT-116) cell lines and found that gossypol induced hyperacetylation of histone protein H3 and/or tubulin within 6 h. Furthermore, incubation with different concentrations of gossypol (5–50 µM) over a time period of 96 h led to a prominent reduction in cellular viability and proliferation of hepatoma (HepG2, Hep3B) and colon carcinoma (HCT-116, HT-29) cells. In-depth analysis of underlying mechanisms showed that gossypol induced apoptosis via caspase activation. For pre-clinical evaluation, toxicity analyses showed toxic effects of gossypol in vitro toward non-malignant primary hepatocytes (PHH), the colon-derived fibroblast cell line CCD-18Co, and the intestinal epithelial cell line CCD 841 CoN at concentrations of ≥5 µM, and embryotoxicity in chicken embryos at ≥2.5 µM. In conclusion, the pronounced inhibitory capacity of gossypol on cancer cells was characterized, and pan-HDACi activity was detected in silico, in vitro, by inhibiting individual HDAC isoenzymes, and on protein level by determining histone acetylation. However, for clinical application, further chemical optimization is required to decrease cellular toxicity.

Hepatic CYP3A4 Enzyme Compensatively Maintains Endogenous Geranylgeranoic Acid Levels in MAOB-Knockout Human Hepatoma Cells.

Geranylgeranoic acid (GGA), developed as a preventive agent against second primary hepatoma, has been reported to be biosynthesized via the mevalonate pathway in human hepatoma-derived cells. Recently, we found that monoamine oxidase B (MAOB) catalyzed the oxidation of geranylgeraniol (GGOH) to produce geranylgeranial (GGal), a direct precursor of endogenous GGA in hepatoma cells, using tranylcypromine, an inhibitor of MAOs, and knockdown by MAOB siRNA. However, endogenous GGA level was unexpectedly unchanged in MAOB-knockout (KO) cells established using the CRISPR-Cas9 system, suggesting that some other latent metabolic pathways maintain endogenous GGA levels in the MAOB-KO cells. Here, we investigated the putative latent enzymes that oxidize GGOH in Hep3B/MAOB-KO cells. First, the broad-specific cytochrome P450 enzyme inhibitors decreased the amount of endogenous GGA in Hep3B/MAOB-KO cells in a dose-dependent manner. Second, among the eight members of cytochrome P450 superfamily that have been suggested to be involved in the oxidation of isoprenols and/or retinol in previous studies, only the CYP3A4 gene significantly upregulated its cellular mRNA level in Hep3B/MAOB-KO cells. Third, a commercially available recombinant human CYP3A4 enzyme was able to oxidize GGOH to GGal, and fourth, the knockdown of CYP3A4 by siRNA significantly reduced the amount of endogenous GGA in Hep3B/MAOB-KO cells. These results indicate that CYP3A4 can act as an alternative oxidase for GGOH when hepatic MAOB is deleted in the human hepatoma-derived cell line Hep3B, and that endogenous GGA levels are maintained by a multitude of enzymes.

Mitochondriotropic antioxidant based on caffeic acid AntiOxCIN4 activates Nrf2-dependent antioxidant defenses and quality control mechanisms to antagonize oxidative stress-induced cell damage

Mitochondria are key organelles involved in cellular survival, differentiation, and death induction. In this regard, mitochondrial morphology and/or function alterations are involved in stress-induced adaptive pathways, priming mitochondria for mitophagy or apoptosis induction. We have previously shown that the mitochondriotropic antioxidant AntiOxCIN4 (100 μM; 48 h) presented significant cytoprotective effect without affecting the viability of human hepatoma-derived (HepG2) cells. Moreover, AntiOxCIN4 (12.5 μM; 72 h) caused a mild increase of reactive oxygen species (ROS) levels without toxicity to primary human skin fibroblasts (PHSF). As Nrf2 is a master regulator of the oxidative stress response inducing antioxidant-encoding gene expression, we hypothesized that AntiOxCIN4 could increase the resistance of human hepatoma-derived HepG2 to oxidative stress by Nrf2-dependent mechanisms, in a process mediated by mitochondrial ROS (mtROS). Here we showed that after an initial decrease in oxygen consumption paralleled by a moderate increase in superoxide anion levels, AntiOxCIN4 led to a time-dependent Nrf2 translocation to the nucleus. This was followed later by a 1.5-fold increase in basal respiration and a 1.2-fold increase in extracellular acidification. AntiOxCIN4 treatment enhanced mitochondrial quality by triggering the clearance of defective organelles by autophagy and/or mitophagy, coupled with increased mitochondrial biogenesis. AntiOxCIN4 also up-regulated the cellular antioxidant defense system. AntiOxCIN4 seems to have the ability to maintain hepatocyte redox homeostasis, regulating the electrophilic/nucleophilic tone, and preserve cellular physiological functions. The obtained data open a new avenue to explore the effects of AntiOxCIN4 in the context of preserving hepatic mitochondrial function in disorders, such as NASH/NAFLD and type II diabetes.

A rapid increase in lysophospholipids after geranylgeranoic acid treatment in human hepatoma-derived HuH-7 cells revealed by metabolomics analysis

Geranylgeranoic acid (GGA) was developed as a preventative agent against second primary hepatoma, and was reported to induce cell death in human hepatoma cells via Toll-like receptor 4 (TLR4)-mediated pyroptosis. We recently reported that GGA is enzymatically biosynthesized from mevalonic acid in human hepatoma-derived HuH-7 cells and that endogenous GGA is found in most rat organs including the liver. An unbiased metabolomics analysis of ice-cold 50% acetonitrile extracts from control and GGA-treated cells was performed in this study to characterize the intracellular metabolic changes in GGA-induced pyroptosis and to analyze their relationship with the mechanism of GGA-induced cell death. The total positive ion chromatograms of the cellular extracts in ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were apparently unchanged after GGA treatment, but an orthogonal partial least squares-discriminant analysis score plot clearly discriminated the intracellular metabolite profiles of GGA-treated cells from that of control cells. S-plot analysis revealed 15 potential biomarkers up-regulated by 24-h GGA treatment according to their variable importance in the projection value of more than 1, and the subsequent metabolomics analysis identified nine of these metabolites as a group of lysophospholipids containing lysophosphatidylcholine with C16:0, C20:4, or C20:3 fatty acids. The possible roles of these lysophospholipids in GGA-induced pyroptosis are discussed.

Age-Dependent Decrease in Hepatic Geranylgeranoic Acid Content in C3H/HeN Mice and Its Oral Supplementation Prevents Spontaneous Hepatoma.

Geranylgeranoic acid (GGA) has been developed as a preventive agent against second primary hepatoma. Recently, GGA was reported to induce cell death in human hepatoma cells via TLR4-mediated pyroptosis. We have reported that GGA is enzymatically biosynthesized from mevalonic acid in human hepatoma-derived cells and that endogenous GGA is found in most organs of rats. In addition, we found that upregulation of endogenous GGA levels by zaragozic acid A (ZAA) induced cell death in human hepatoma-derived cells. Therefore, we investigated the age-related changes in hepatic GGA and the possibility of suppressing hepatocarcinogenesis by GGA supplementation using male C3H/HeN mice that spontaneously develop hepatoma. We measured endogenous GGA and mRNA of monoamine oxidase (BMAOB), a key enzyme of GGA biosynthesis, in the liver of male C3H/HeN mice aged 6–93 weeks. We also tried suppressing spontaneous hepatocarcinogenesis by a single administration of GGA to C3H/HeN mice. Hepatic GGA content and Maob mRNA expression level age-dependently decreased in male C3H/HeN mice; some of which produced spontaneous hepatoma in 2 years. A single oral administration of GGA at 11 months of age significantly prevented hepatoma in terms of the number and weight of tumors per mouse at 24 months. Oral supplementation with GGA or geranylgeraniol significantly increased endogenous hepatic GGA contents dose-dependently; and ZAA dramatically upregulated hepatic GGA. In this study; we found an age-dependent decrease in hepatic endogenous GGA in male C3H/HeN mice and efficient prevention of spontaneous hepatoma by a single administration of GGA at 11 months of age.

A Novel Small Molecule Inhibits Hepatitis C Virus Propagation in Cell Culture.

ABSTRACTHepatitis C virus (HCV) can cause acute and chronic infection that is associated with considerable liver-related morbidity and mortality. In recent years, there has been a shift in the treatment paradigm with the discovery and approval of agents that target specific proteins vital for viral replication. We employed a cell culture-adapted strain of HCV and human hepatoma-derived cells lines to test the effects of our novel small-molecule compound (AO13) on HCV. Virus inhibition was tested by analyzing RNA replication, protein expression, and virus production in virus-infected cells treated with AO13. Treatment with AO13 inhibited virus spread in cell culture and showed a 100-fold reduction in the levels of infectious virus production. AO13 significantly reduced the level of viral RNA contained within cell culture fluids and reduced the cellular levels of HCV core protein, suggesting that the compound might act on a late step in the viral life cycle. Finally, we observed that AO13 did not affect the release of infectious virus from infected cells. Docking studies and molecular dynamics analyses suggested that AO13 might target the NS5B RNA polymerase, however, real-time RT-PCR analyses of cellular levels of HCV RNA showed only an ∼2-fold reduction in viral RNA levels in the presence of AO13. Taken together, this study revealed that AO13 showed consistent, but low-level antiviral effect against HCV, although the mechanism of action remains unclear.IMPORTANCE The discovery of curative antiviral drugs for a chronic disease such as HCV infection has encouraged drug discovery in the context of other viruses for which no curative drugs currently exist. Since we currently face a novel virus that has caused a pandemic, the need for new antiviral agents is more apparent than ever. We describe here a novel compound that shows a modest antiviral effect against HCV that could serve as a lead compound for future drug development against other important viruses such as SARS-CoV-2.

Docosahexaenoic Acid Esters of Hydroxy Fatty Acid Is a Novel Activator of NRF2.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with interesting physiological functions in mammals. Despite their structural diversity and links with nuclear factor erythroid 2-related factor 2 (NRF2) biosynthesis, FAHFAs are less explored as NRF2 activators. Herein, we examined for the first time the synthetic docosahexaenoic acid esters of 12-hydroxy stearic acid (12-DHAHSA) or oleic acid (12-DHAHOA) against NRF2 activation in cultured human hepatoma-derived cells (C3A). The effect of DHA-derived FAHFAs on lipid metabolism was explored by the nontargeted lipidomic analysis using liquid chromatography-mass spectrometry. Furthermore, their action on lipid droplet (LD) oxidation was investigated by the fluorescence imaging technique. The DHA-derived FAHFAs showed less cytotoxicity compared to their native fatty acids and activated the NRF2 in a dose-dependent pattern. Treatment of 12-DHAHOA with C3A cells upregulated the cellular triacylglycerol levels by 17-fold compared to the untreated group. Fluorescence imaging analysis also revealed the suppression of the degree of LDs oxidation upon treatment with 12-DHAHSA. Overall, these results suggest that DHA-derived FAHFAs as novel and potent activators of NRF2 with plausible antioxidant function.

Exploratory Data Analysis of Cell and Mitochondrial High-Fat, High-Sugar Toxicity on Human HepG2 Cells.

Non-alcoholic steatohepatitis (NASH), one of the deleterious stages of non-alcoholic fatty liver disease, remains a significant cause of liver-related morbidity and mortality worldwide. In the current work, we used an exploratory data analysis to investigate time-dependent cellular and mitochondrial effects of different supra-physiological fatty acids (FA) overload strategies, in the presence or absence of fructose (F), on human hepatoma-derived HepG2 cells. We measured intracellular neutral lipid content and reactive oxygen species (ROS) levels, mitochondrial respiration and morphology, and caspases activity and cell death. FA-treatments induced a time-dependent increase in neutral lipid content, which was paralleled by an increase in ROS. Fructose, by itself, did not increase intracellular lipid content nor aggravated the effects of palmitic acid (PA) or free fatty acids mixture (FFA), although it led to an up-expression of hepatic fructokinase. Instead, F decreased mitochondrial phospholipid content, as well as OXPHOS subunits levels. Increased lipid accumulation and ROS in FA-treatments preceded mitochondrial dysfunction, comprising altered mitochondrial membrane potential (ΔΨm) and morphology, and decreased oxygen consumption rates, especially with PA. Consequently, supra-physiological PA alone or combined with F prompted the activation of caspase pathways leading to a time-dependent decrease in cell viability. Exploratory data analysis methods support this conclusion by clearly identifying the effects of FA treatments. In fact, unsupervised learning algorithms created homogeneous and cohesive clusters, with a clear separation between PA and FFA treated samples to identify a minimal subset of critical mitochondrial markers in order to attain a feasible model to predict cell death in NAFLD or for high throughput screening of possible therapeutic agents, with particular focus in measuring mitochondrial function.

Insertion of a Synthetic Switch Into Insulin Provides Metabolite-Dependent Regulation of Hormone-Receptor Activation

Insulin signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to large-scale structural reorganization. To test the coupling between insulin’s “opening” and receptor activation, we inserted an artificial ligand-dependent switch into insulin. Ligand binding disrupts an internal tether designed to stabilize the hormone’s native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the receptor. Similarly, metabolite-regulated signaling was not observed in control studies of (i) an unmodified insulin analog or (ii) an analog containing a diol sensor in the absence of internal tethering. Although as expected CD-detected secondary structure was unaffected by ligand binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and holoreceptor signaling. In addition to this basic finding, our results provide proof of principle for a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a “smart” insulin analog designed to mitigate risk of hypoglycemia in the treatment of diabetes mellitus.

Identification of Characteristic Genomic Markers in Human Hepatoma HuH-7 and Huh7.5.1-8 Cell Lines.

The human hepatoma-derived HuH-7 cell line and its derivatives (Huh7.5 and Huh7.5.1) have been widely used as a convenient experimental substitute for primary hepatocytes. In particular, these cell lines represent host cells suitable for propagating the hepatitis C virus (HCV) in vitro. The Huh7.5.1-8 cell line, a subline of Huh7.5.1, can propagate HCV more efficiently than its parental cells. To provide genomic information for cells’ quality control, we performed whole-genome sequencing of HuH-7 and Huh7.5.1-8 and identified their characteristic genomic deletions, some of which are applicable to an in-house test for cell authentication. Among the genes related to HCV infection and replication, 53 genes were found to carry missense or loss-of-function mutations likely specific to the HuH-7 and/or Huh7.5.1-8. Eight genes, including DDX58 (RIG-I), BAX, EP300, and SPP1 (osteopontin), contained mutations observed only in Huh7.5.1-8 or mutations with higher frequency in Huh7.5.1-8. These mutations might be relevant to phenotypic differences between the two cell lines and may also serve as genetic markers to distinguish Huh7.5.1-8 cells from the ancestral HuH-7 cells.

RORα phosphorylation by casein kinase 1α as glucose signal to regulate estrogen sulfation in human liver cells.

Estrogen sulfotransferase (SULT1E1) metabolically inactivates estrogen and SULT1E1 expression is tightly regulated by multiple nuclear receptors. Human fetal, but not adult, livers express appreciable amounts of SULT1E1 protein, which is mimicked in human hepatoma-derived HepG2 cells cultured in high glucose (450 mg/dl) medium. Here, we have investigated this glucose signal that leads to phosphorylation of nuclear receptor RORα (NR1F1) at Ser100 and the transcription mechanism by which phosphorylated RORα transduces this signal to nuclear receptor HNF4α, activating the SULT1E1 promoter. The promoter is repressed by non-phosphorylated RORα which binds a distal enhancer (−943/−922 bp) and interacts with and represses HNF4α-mediated transcription. In response to high glucose, RORα becomes phosphorylated at Ser100 and reverses its repression of HNF4α promoter activation. Moreover, the casein kinase CK1α, which is identified in an enhancer-bound nuclear protein complex, phosphorylates Ser100 in in vitro kinase assays. During these dynamic processes, both RORα and HNF4α remain on the enhancer. Thus, RORα utilizes phosphorylation to integrate HNF4α and transduces the glucose signal to regulate the SULT1E1 gene in HepG2 cells and this phosphorylation-mediated mechanism may also regulate SULT1E1 expressions in the human liver.