HepG2 Cell Model Research Articles

Evaluation of the Inhibitory Effects of Genipin on the Fluoxetine-Induced Invasive and Metastatic Model in Human HepG2 Cells.

Metastasis of hepatocellular carcinoma (HCC) is usually unrecognized before any pathological examination, resulting in time-taking treatment and poor prognosis. As a consequence, HCC patients usually show symptoms of depression. In order to suppress such psychiatric disorders and to facilitate better treatment outcome, antidepressants are prescribed. Up to present, information about the effect of antidepressants on HCC is still lacking. Therefore, we chose fluoxetine (FXT), one of the top five psychiatric prescriptions in the United States, together with the HepG2 cell model to explore its effect on HCC. Our study found that FXT (5 µM) increased the migratory distance of HepG2 cells by a factor of nearly 1.7 compared to control. In addition, our study also investigated the effect of genipin (GNP), which is an active compound from Gardenia jasminoides Ellis fruit (family Rubiaceae), on the FXT-induced HepG2 cells. Our study found that 30 and 60 µM GNP reduced the migratory distance by 42% and 74% respectively, compared to FXT treatment alone. Furthermore, we also found that FXT upregulated matrix metalloproteinases (MMPs) genes, increased the protein expression of MMPs, urokinase-type plasminogen activator (uPA), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein 1 (AP-1), phosphorylated mitogen-activated protein kinase (P-p38), phosphorylated protein kinase B (P-Akt), downregulated tissue inhibitor metalloproteinases (TIMPs) genes and decreased the TIMPs proteins expression whereas, GNP fully counteracted the action of FXT. Conclusively, this study has provided valuable information regarding the possible molecular mechanisms through which FXT affects the metastatic invasiveness of HepG2 cells and evidences to support that GNP counteracts such effect via the same molecular mechanisms.

Flavonoids, phenolic acids, carotenoids and antioxidant activity of fresh eating citrus fruits, using the coupled in vitro digestion and human intestinal HepG2 cells model

With in-vitro digestion and human intestinal HepG2 cells, we analyzed the bioaccessibility and cell uptake of phytochemicals and determined the cellular antioxidant capacity (CAA) of fresh eating citrus fruits. The results showed that CAA of citrus fruits was higher in digesta than in extracts, and the CAA is strongly correlated with naringenin and beta-carotene uptake (p < 0.05). During in vitro digestion, vanillic acid and p-coumaric decreased, and ferulic acid increased in all citrus fruits significantly (p < 0.05); other phytochemicals varied among the fruits. During uptake, hydroxybenzoic acids, hesperidin, narirutin, naringenin and neohesperidin were detected in cells, Zeaxanthin, lutein, beta-cryptoxanthin and beta-carotene could be detected in the citrus varieties except for pummel, but hydroxycinnamic acids and hesperitin were not detected in cells. This work provides insights into the bioaccessibility and cell uptake of phytochemicals and cellular antioxidant activity of fresh eating citrus fruits.

Novel hepatoprotective role of Leonurine hydrochloride against experimental non-alcoholic steatohepatitis mediated via AMPK/SREBP1 signaling pathway

Background and aimsNon-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome and is characterized by steatosis, inflammation, and fibrosis. We aim to characterize the hepatoprotective effects of Leonurine hydrochloride (LH) and the possible pathway in a cell and rodent model of diet-induced steatohepatitis (NASH). MethodsFor in vitro studies, Palmitic acid (PA) and free fatty acid (FFA) induced HepG2 and HL7702 steatosis cell models were used. For in vivo studies, NASH was induced by feeding mice MCD diet. These mice received either placebo or LH at three different doses (50、100、200 mg/kg/day) for 6 weeks. Histological staining’s, and commercially available kits for ALT and AST and hepatic contents of TG, TC, MDA, SOD, and GSH were used to assess NASH. Furthermore, relative liver protein and gene expression levels were determined by Western Blot and qPCR, respectively. ResultsAfter establishing NASH models, LH treatment improved lipid accumulation, hepatic contents of TG, TC, and expression levels of ALT and AST in dose-dependent manner. Also, LH improved MDA, SOD, and GSH expression levels. The results of RT-PCR and Western blotting showed that LH upregulated the expression of AMPK phosphorylation and downregulated SREBP-1c and its target genes expression level. ConclusionsOur data reveal the promising role of Leonurine hydrochloride in the prevention and treatment of NASH, in vitro and in vivo. This effect may be partially mediated by the AMPK/SREBP1 pathway. These findings provide a novel therapeutic target for the clinical treatment of NASH.

Effects of sera of rats fed with Huganqingzhi tablets on endoplasmic reticulum stress in a HepG2 cell model of nonalcoholic fatty liver disease

To investigate the effects of sera from rats fed with Huganqingzhi tablets (HGT) on endoplasmic reticulum (ER) stress in a steatotic hepatocyte model of free fatty acids (FFAs)-induced nonalcoholic fatty liver disease (NAFLD) and explore the possible mechanism. FFAs prepared by mixing oleic acid and palmitic acid at the ratio of 2:1. HepG2 cells were treated with the sera from rats fed with low-, moderate-or high-dose HGT (HGT sera) or sera of rats fed with fenofibrate (fenofibrate sera), followed by treatment with 1 mmol/L FFAs for 24 h to induce hepatic steatosis. Oil red O staining was used to observe the distribution of lipid droplets in the cells. The biochemical parameters including triglyceride (TG), lactated hydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using a commercial kit. The morphological changes of the ER in the cells were observed using transmission electron microscopy. The protein/mRNA expressions of ER stress-related signal molecules including GRP78, PERK, p-PERK, ATF6, ATF4, CASPASE-12, CHOP, XBP-1, PKC, and p-PKC-δ were detected using Western blotting and/or quantitative real-time PCR (qRT-PCR). The changes in the protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP were also detected in cells with transient transfection of PKC-δ siRNA for PKC-δ knockdown. Compared with the control cells, the cells treated with FFAs showed significantly increased levels of TG, AST, and ALT (P &lt; 0.05). Compared with FFAs-treated cells, the cells pretreated with HGT sera or fenofibrate sera all showed significantly decreased TG, AST and ALT levels (P &lt; 0.05), reduced accumulation of the lipid droplets (P &lt; 0.05), and lowered protein or mRNA expression levels of GRP78, p-PERK, ATF6, ATF4, CHOP, CASPASE-12, XBP-1 and p-PKC-δ (P &lt; 0.05). PKC-δ knockdown caused significantly reduced protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP in the cells with FFA-induced hepatic steatosis (P &lt; 0.001); treatment with high-dose HGT serum more significantly reduced the expressions of GRP78 (P &lt; 0.001) and P-PERK (P &lt; 0.01) in FFAs-induced cells with PKC-δ knockdown. HGT serum can effectively prevent FFAs-induced steatosis in HepG2 cells by alleviating ER stress, in which PKC-δ may act as an important target.

BH3 mimetics derived from Bim-BH3 domain core region show PTP1B inhibitory activity

A series of our previously described BH3 peptide mimetics derived from Bim-BH3 domain core region were found to exhibit weak to potent PTP1B binding affinity and inhibitory activities via target-based drug screening. Among these compounds, a 12-aa Bim-BH3 core sequence peptide conjugated to palmitic acid (SM-6) displayed good PTP1B binding affinity (KD = 8.38 nmol/L), inhibitory activity (IC50 = 1.20 μmol/L) and selectivity against other PTPs (TCPTP, LAR, SHP-1 and SHP-2). Furthermore, SM-6 promoted HepG2 cell glucose uptake and inhibited the expression of PTP1B, indicating that SM-6 could improve the insulin resistance effect in the insulin-resistant HepG2 cell model. These results may indicate a new direction for the application of BH3 peptide mimetics and promising PTP1B peptide inhibitors could be designed and developed based on SM-6.

Lysine at position 329 within a C-terminal dilysine motif is crucial for the ER localization of human SLC35B4.

SLC35B4 belongs to the solute carrier 35 (SLC35) family whose best-characterized members display a nucleotide sugar transporting activity. Using an experimental model of HepG2 cells and indirect immunofluorescent staining, we verified that SLC35B4 was localized to the endoplasmic reticulum (ER). We demonstrated that dilysine motif, especially lysine at position 329, is crucial for the ER localization of this protein in human cells and therefore one should use protein C-tagging with caution. To verify the importance of the protein in glycoconjugates synthesis, we generated SLC35B4-deficient HepG2 cell line using CRISPR-Cas9 approach. Our data showed that knock-out of the SLC35B4 gene does not affect major UDP-Xyl- and UDP-GlcNAc-dependent glycosylation pathways.

Constitutive androstane receptor weakens the induction of panaxytriol on CYP3A4 by repressing the activation of pregnane X receptor

Nuclear receptors pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3) play a vital role in regulating CYP3A4. Our previous studies have demonstrated that panaxytriol (PXT) upregulates the expression of CYP3A4 via the PXR regulatory pathway. This study aimed to explore how CAR mediates the regulation of CYP3A4 in the presence of PXT using HepG2 cell, hCAR-overexpressing HepG2 cell and hCAR-silenced HepG2 cell models. In HepG2 cells, PXT induced the expression of CYP3A4 in a concentration-dependent manner (10–80 μM) and the high concentration of PXT (80 μM) upregulated the expression of CAR. The concentrations of PXT (10–40 μM) had no impact on the expression of CAR, but could significantly induce the expression of CYP2B6 target gene by activating CAR. The dual-luciferase reporter gene assay also showed that CAR-mediated CYP3A4 luciferase activity can be promoted by 80 μM of PXT (1.54-fold), while 5, 10, 20, and 40 μM of PXT had no influence on CAR-mediated CYP3A4 luciferase activity. In hCAR-overexpressing HepG2 cells, PXT concentrations (10–40 μM) that significantly induced PXR and CYP3A4 in HepG2 cells had no impact on the expression of CYP3A4, CAR and PXR, whereas a high concentration of PXT (80 µM) could weakly induce the mRNA and protein levels of CAR and CYP3A4. Moreover, the expression of PXR and CYP3A4 in hCAR-silenced HepG2 cells was markedly elevated compared with the blank control or with normal HepG2 cells treated with 10–80 μM of PXT. In conclusion, CAR significantly weakens the ability of PXT to induce CYP3A4 expression by repressing the activation of PXR. There may be a cross-talk mechanism between PXR and CAR on the regulation of CYP3A4 in the presence of PXT. Additionally, a high concentration of PXT (80 μM) induced CYP3A4 via the CAR regulatory pathway.

Stable Isotope-Labeled Lipidomics to Unravel the Heterogeneous Development Lipotoxicity.

Non-alcoholic fatty liver disease (NAFLD) as a global health problem has clinical manifestations ranging from simple non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH), cirrhosis, and cancer. The role of different types of fatty acids in driving the early progression of NAFL to NASH is not understood. Lipid overload causing lipotoxicity and inflammation has been considered as an essential pathogenic factor. To correlate the lipid profiles with cellular lipotoxicity, we utilized palmitic acid (C16:0)- and especially unprecedented palmitoleic acid (C16:1)-induced lipid overload HepG2 cell models coupled with lipidomic technology involving labeling with stable isotopes. C16:0 induced inflammation and cell death, whereas C16:1 induced significant lipid droplet accumulation. Moreover, inhibition of de novo sphingolipid synthesis by myriocin (Myr) aggravated C16:0 induced lipoapoptosis. Lipid profiles are different in C16:0 and C16:1-treated cells. Stable isotope-labeled lipidomics elucidates the roles of specific fatty acids that affect lipid metabolism and cause lipotoxicity or lipid droplet formation. It indicates that not only saturation or monounsaturation of fatty acids plays a role in hepatic lipotoxicity but also Myr inhibition exasperates lipoapoptosis through ceramide in-direct pathway. Using the techniques presented in this study, we can potentially investigate the mechanism of lipid metabolism and the heterogeneous development of NAFLD.

Antioxidant properties of chitosan-(poly)nitroxides under induced oxidative stress

The cytotoxicity and antioxidant properties of water-soluble (at physiological pH) chitosan- (poly)nitroxides (CPNs) containing pyrroline-based nitroxide and having different molecular weights of the polysaccharide backbone were studied on normal (Vero) and tumor (HepG2) cell models. The compounds were found to exhibit low cytotoxicity and to effectively reduce the level of reactive oxygen species (ROS) generated upon the breakdown of tert-butyl hydroperoxide in normal cells. In tumor cells, the CPNs have little effect on the ROS level under the oxidative stress. The results show that CPNs also represent a promising platform for the development of cell delivery systems for biologically active compounds.

Polysaccharides from pineapple pomace: new insight into ultrasonic-cellulase synergistic extraction and hypoglycemic activities

This study determined the optimal extraction conditions for ultrasonic-cellulase synergistic extraction of polysaccharides from pineapple pomace (PPP) using Plackett–Burman design and response surface methodology. The optimal hydrolysis temperature, ratio of material to water, pH value, hydrolysis time, ultrasonic power and the additive quantity of cellulase were 50 °C, 1:45 g/mL, 6.0, 100 min, 160 W and 2.0%, respectively, giving a extraction yield of 1.10 ± 0.03%. PPP was further isolated and purified by DEAE-52 cellulose and Sephadex G-100 chromatography columns, revealing four main elution peaks, named PPF0, PPF1, PPP2 and PPF3, were obtained. The molecular weight, monosaccharide compositions, structural features and appearance morphology of polysaccharide fractions (PPFs) were analyzed by high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC), UV spectroscopy, fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). Furthermore, the hypoglycemic activities of PPFs with different concentrations were also investigated by insulin resistance HepG2 cells model in vitro. Results showed that PPF0, PPF1, PPF2 and PPF3 were composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose with molecular weight of 6.71 × 104, 1.11 × 104, 2.22 × 104 and 5.1 × 103 Da, respectively. All of them could alleviate the development of insulin resistance HepG2 cells with a dose-dependent relationship. The glucose consumption increased 46.4%, 50.5%, 82.1% and 53.6%; 86.8%, 81.6%, 86.8% and 84.2% at the concentration of 20 μg/mL, respectively, without or with insulin. These results suggested that PPFs can be explored as a potential hypoglycemic agent in biomedical and functional food.

Differentiated hypoglycemic effects of baicalin, berberine and puerarin on insulin-resistance HepG2 cells

To investigate the hypoglycemic effects of baicalin, berberine, puerarin and liquiritin on the insulin resistance (IR) cells. The IR model of HepG2 cells was established by treatment with insulin and dexamethasone for 48 h. Glucose uptake, glycogen content and cell viability were detected with different concentrations of baicalin, berberine, puerarin, liquiritin in IR-HepG2 cells. Compared with IR model group, all of intervened groups significantly increased the glucose consumption, except for liquiritin groups and 1 μmol·L⁻¹ baicalin group. Moreover, 10, 20, 50 μmol·L⁻¹ baicalin, 5, 10, 20, 50 μmol·L⁻¹ berberine and 40, 80, 160 μmol·L⁻¹ puerarin significantly elevated glycogen content in IR-HepG2 cells. Liquiritin did not show obvious hypoglycemic effect. Compared with normal group, the mRNA expression levels of GLUT1 and GLUT4 were decreased in IR-HepG2 cells according to qPCR results. 5, 20 μmol·L⁻¹ berberine decreased the mRNA expression level of GLUT1 in IR-HepG2 cells, whereas 20, 40, 80 μmol·L⁻¹ puerarin significantly elevated the mRNA expression level of GLUT1. Moreover, 10, 20, 50 μmol·L⁻¹ baicalin and 20 μmol·L⁻¹ berberine increased the mRNA expression level of GLUT4. Whereas, 40, 80 μmol·L⁻¹ puerarin decreased the mRNA expression level of GLUT4. Western blot results suggested that 10, 20, 50 μmol·L⁻¹ baicalin significantly increased the protein expressions of GLUT2 and GLUT4, whereas 20, 40, 80 μmol·L⁻¹ puerarin significantly up-regulated GLUT1 and GLUT2 proteins. In addition, 20 μmol·L⁻¹ berberine increased the protein expressions of GLUT2 and GLUT4, whereas 10 μmol·L⁻¹ berberine up-regulated GLUT4 expression. The results preliminarily suggested that baicalin, berberine and puerarin have differentiated hypoglycemic effects, which accelerate glucose transport, increase glycogen synthesis, regulate glucose metabolism and improve hepatic IR.

Hypolipidemic effect of SIPI-7623, a derivative of an extract from oriental wormwood, through farnesoid X receptor antagonism

Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. As a metabolic regulator, FXR plays key roles in bile acid and cholesterol metabolism and lipid and glucose homeostasis. Therefore, FXR is a potential drug target for several metabolic syndromes, especially those related to lipidemia disorders. In the present study, we identified small molecule SIPI-7623, a derivative of an extract from Oriental wormwood (Artemisia capillaris), and found that it specifically upregulated the expression of cholesterol-7-alpha-hydroxylase (CYP7A1), downregulated the expression of sterol-regulatory element-binding protein 1c (SREBP-1c) in the liver, and inhibited the expression of ileal bile acid binding-protein (IBABP) in the ileum of rats. We found that inhibition of FXR by SIPI-7623 decreased the level of cholesterol and triglyceride. SIPI-7623 reduced the levels of cholesterol and triglyceride in in vitro HepG2 cell models, ameliorated diet-induced atherosclerosis, and decreased the serum lipid content on rats and rabbits model of atherosclerosis in vivo. Furthermore, SIPI-7623 decreased the extent of atherosclerotic lesions. Our resutls demonstrated that antagonism of the FXR pathway can be employed as a therapeutic strategy to treat metabolic diseases such as hyperlipidemia and atherosclerosis. In conclusion, SIPI-7623 could be a promising lead compound for development of drugs to treat hyperlipidemia and atherosclerosis.

Hepatitis B virus upregulates host microRNAs that target apoptosis-regulatory genes in an in vitro cell model

Chronic hepatitis B (CHB) infection increases the risk of developing severe liver disease including cirrhosis and hepatocellular carcinoma (HCC). As microRNAs may modulate host – virus interactions, we here investigated if hepatitis B virus (HBV) infection modulate microRNA expression using an in vitro HepG2 cell model system with inducible HBV replication. We found that HBV replication was associated with upregulation of miR-192-5p, miR-194-5p and miR-215-5p, of which miR-192-5p and miR-215-5p have identical seed sequences. Bioinformatics analyses revealed a significant enrichment of potential target genes involved in apoptosis signaling of all three microRNAs. In line with this, transfection with a mimic of miR-192-5p suppressed the protein level of pro-apoptotic BIM and reduced endoplasmic reticulum (ER) stress-induced apoptosis in HepG2 cells. In contrast, transfection with a mimic of miR-194-5p downregulated the anti-apoptotic proteins SODD and cFLIP, and sensitized HepG2 cells to both ER stress- and cytokine-induced apoptosis. In conclusion, our study suggests that HBV upregulates the expression of miR-192-5p and miR-194-5p in the host cell. These microRNAs target important apoptosis-regulatory proteins, and may thus contribute to the development of HBV-related liver disease.

13. Picroside ii reduces lipid accumulation, oxidative stress and mitochondrial dysfunction in in vitro NAFLD HePG2 cell model

13. Picroside ii reduces lipid accumulation, oxidative stress and mitochondrial dysfunction in in vitro NAFLD HePG2 cell model

Curcumin inhibits liver cancer by inhibiting DAMP molecule HSP70 and TLR4 signaling.

Curcumin has been revealed to inhibit liver cancer, however, no studies have reported that the mechanism of curcumin's action on liver cancer is related to damage-associated molecular pattern (DAMP) molecules heat shock protein70 (HSP70) and the toll-like receptor 4 (TLR4) signaling. This study aimed to investigate whether the activation of TLR4 signaling by HSP70 could be inhibited by curcumin, thus investigating the possible mechanism of curcumin in the inhibition of liver cancer. Western blotting was used to evaluate the expression of the HSP70 and TLR4 in HepG2 cells and ELISA was used to detect the concentration of HSP70 in cell culture medium. A thermal tolerance HepG2 (HepG2TT) cell model was established to simulate HSP70 accumulation in the microenvironment. A certain concentration of curcumin was co-cultured with HepG2 and HepG2TT cells to observe the changes of HSP70 and TLR4. Our results revealed that heat stress significantly increased the expression of extracellular HSP70 (eHSP70) and TLR4 (P<0.01), but significantly reduced the expression of intracellular HSP70 (P<0.01). Curcumin inhibited proliferation, invasion, and metastasis of HepG2 cells, caused cells to remain in the DNA Sphase, promoted apoptosis, and significantly reduced intracellular HSP70, eHSP70 and TLR4 levels of HepG2TT cells. Following the removal of curcumin, eHSP70 increased again. In summary, our results demonstrated that the antitumor effect of curcumin was related to the inhibition HSP70-TLR4 signaling.

MiRNA-17 encoded by the miR-17-92 cluster increases the potential for steatosis in hepatoma cells by targeting CYP7A1

BackgroundThe miRNA cluster miR-17-92 is known to act as an oncogene in various cancers. Members of this cluster were also found to be involved in some other pathological process, such as steatosis, which is a pivotal event in the initiation and progression of nonalcoholic fatty liver disease (NAFLD). This study aimed to explore whether miR-17, one of the most functional miRNAs in the miR-17-92 family, participates in the process of steatosis in hepatoma cells.MethodsWe developed both a miR-17-expressing transgenic mouse model and a miR-17-expressing HepG2 cell model, the latter was established via stable transfection. Real-time PCR and western blot were applied to measure the expression levels of miR-17 and the potential target gene CYP7A1. The luciferase assay was used to confirm direct binding of miR-17 and CYP7A1. The oleic acid induction assay and Oil-Red-O staining were performed to support the determination of steatotic changes in HepG2 cell.ResultsExtensive steatotic changes were observed in the livers of transgenic mice. Fewer were seen in the wild-type animals. CYP7A1 was confirmed as a target gene of miR-17, and the expression of CYP7A1 was found to be negatively regulated in both the transgenic mice liver cells and the miR-17-expressing HepG2 cells. CYP7A1 was found to participate in miR-17-induced steatosis, as its repressed expression in miR-17 HepG2 cells exacerbated steatotic change. Re-introduction of CYP7A1 into miR-17 HepG2 cell partially alleviated steatosis.ConclusionsmiR-17 is a novel regulator of CYP7A1 signaling in hepatic lipid metabolism, suggesting a potential therapeutic approach for fatty liver.

The Role of Lipocalin‐2 (LCN2) and High Mobility Box Group 1 (HMGB1) in Acetaminophen‐induced Acute Liver Failure

Acetaminophen (APAP) overdose is the most common cause of Acute Liver Failure (ALF). Following APAP overdose, toxicity is initiated by the conversion of APAP to its reactive metabolic N‐acetyl‐p‐benzoquinone imine (NAPQI) by CYP2E1/CYP3A4. NAPQI binds covalently to mitochondrial proteins forming 3‐(cysteine‐S‐yl)‐acetaminophen (APAP‐Cys). This generates initial hepatotoxicity in the form of centrilobular necrosis and sterile inflammation. Lipocalin‐2 (LCN2), is an acute‐phase innate immune protein, upregulated during tissue injury in various organs including the liver. Recently we have demonstrated that LCN2 is pro‐inflammatory and elevated during APAP overdose in mouse model. Interestingly, LCN2 knockout (LCN2 KO) mice exhibit protection from APAP overdose compared to its wild type (WT) littermates. During APAP overdose there is necrosis of hepatocytes, these hepatocytes release Damage Associated Molecular Patterns (DAMPS) that further exacerbate the inflammatory process. One such DAMP is High Mobility Group Box 1 (HMGB1). However, the role of LCN2 and HMGB1 in APAP overdose has not yet been investigated.Present study investigates the molecular mechanism of LCN2 during the progression of acute liver injury in APAP overdose model. We hypothesize that during APAP induced ALF, LCN2 induces the expression and translocation of HMGB1 which in turn mediates inflammation and progression of injury. An in vitro model of HepG2 cells as well as an in vivo model of WT and LCN2 KO mice will be utilized to test our hypothesis. HepG2 cells will be treated with recombinant LCN2 to investigate whether exogenous LCN2 can cause induction and translocation of HMGB1. Translocation of HMGB1 will be assessed by immunofluorescence staining. Serum and tissues samples of WT and LCN2 KO mice subjected to APAP overdose will be assessed for expression of HMGB1. APAP‐cys formation will be measured in WT and LCN2 KO using the High‐Performance Liquid Chromatography method to ensure comparable bioactivation based injury between the WT and LCN2 KO mice. Lastly, histopathology will be performed on liver samples from both groups to assess liver pathology. It is anticipated that LCN2 mediates progression of liver injury in APAP overdose via HMGB1.Support or Funding InformationPhiladelphia College of Osteopathic Medicine‐Division of ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel metformin‐methylglyoxal imidazolinone metabolite (IMZ) sensitizes cells to insulin; a potential role in alleviating T2DM complications

Reactive dicarbonyls, such as methylglyoxal (MG), are elevated in type‐two diabetes mellitus (T2DM) patients. These endogenous electrophiles covalently modify proteins, which may contribute to diabetic complications. The T2DM first‐line therapy, metformin (MF), significantly reduces adverse diabetic endpoints and mortality more effectively than other anti‐hyperglycemic agents, the mechanism(s) of which remain unclear. We previously identified and characterized the product of the MF and MG reaction as a novel five‐membered imidazolinone (IMZ) metabolite. IMZ was detected via LC/MS in all MF‐treated T2DM patients, and increased MF urinary levels directly correlated with elevations in urinary IMZ. Scavenging of MG by MF represents a possible alternative mechanism of MF drug efficacy, in addition to its antigluconeogenesis properties. Imidazoline receptor subtypes (I1R, I2R, and I3R) are novel targets for drug development in disorders associated with T2DM because they are involved in insulin sensitization, insulin secretion, and glucose homeostasis. Thus, we examined the ability of IMZ to modulate insulin‐mediated cell signaling pathways via western blot in PC12 cells, which express high levels of I1R and lack the α2‐adrenergic receptor, which many I1R agonists also activate. Combination treatment of insulin and IMZ at physiologically relevant concentrations increased AKT and ERK1/2 phosphorylation above levels seen with insulin treatment alone. This potentiation was not observed in the presence of I1R antagonists. Moreover, IMZ restores insulin sensitivity in an insulin resistant HEPG2 cell model. The results revealed that IMZ may enhance insulin action in the insulin‐dependent AKT and ERK1/2 pathways through I1R activation. Preliminary in vivo pharmacokinetic studies show that IMZ (IP; 10mg/kg and 20mg/kg) is rapidly absorbed, with an elimination half‐life of 12–16.5 minutes. In addition, IMZ enhanced hepatic pAKT 40 minutes after the 10mg/kg dose. In summary, the formation of IMZ in T2DM patients provides evidence that MF scavenges MG, potentially reducing detrimental protein modifications. This property of MF may play a role in the reduction of diabetic complications and represent a potential alternative mechanism of MF drug efficacy. Further, IMZ itself may have the ability to enhance insulin action, suggesting possible clinical utility in the treatment of T2DM.Support or Funding InformationR24DK0090958, T32ES007091, P30ES006694This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bioavailability and metabolism of rosemary infusion polyphenols using Caco-2 and HepG2 cell model systems.

Rosmarinus officinalis is an aromatic plant used in folk medicine as a result of the therapeutic properties associated with its phenolic composition, being rich in rosmarinic acid (RA) and caffeic acid (CA). To better understand the bioactivity of these compounds, their absorption and metabolism were assessed in human Caco-2 and HepG2 cells, as small intestine and liver models, respectively, using RA and CA standards, as well as a rosemary infusion and ferulic acid (FA). Test compounds were partially up-taken and metabolized by Caco-2 and HepG2 cells, although a higher metabolization rate was observed after hepatic incubation compared to intestinal incubation. CA was the compound best absorbed followed by RA and FA, showing metabolites percentages of 30.4%, 11.8% and 4.4% in Caco-2 and 34.3%, 10.3% and 3.2% in HepG2 cells, respectively. RA in the rosemary infusion showed improved bioavailability compared to pure RA. Methyl derivatives were the main metabolites detected for CA and RA after intestinal and hepatic metabolism, followed by methyl-glucuronidates and glucuronidates. RA was also minimally hydrolyzed into CA, whereas FA only was glucuronidated. Rosemary polyphenols followed the same biotransformation pathways as the standards. In addition, phase II derivatives of luteolin were observed. Rosemary polyphenols are partially metabolized in both the intestine and liver. © 2018 Society of Chemical Industry.

Hypolipidemic activity and mechanisms of the total phenylpropanoid glycosides from Ligustrum robustum (Roxb.) Blume by AMPK-SREBP-1c pathway in hamsters fed a high-fat diet.

The aim of this study was to evaluate the hypolipidemic effect and mechanisms of total phenylpropanoid glycosides extracted from Ligustrum robustum (Roxb.) Blume (LRTPG) in hamsters fed a high-fat diet and to discover bioactive components in HepG2 cell model induced by oleic acid. LRTPG of high (1.2g/kg), medium (0.6g/kg), and low (0.3g/kg) doses was administrated daily for 21 consecutive days in hamsters. We found that in hamsters fed a high-fat diet, LRTPG effectively reduced the concentrations of plasma triglycerides (TG), free fatty acid, total cholesterol, low-density lipoprotein cholesterol, and hepatic TG and total cholesterol. And the compounds acteoside, ligupurpuroside A, ligupurpuroside C, and ligupurpuroside D significantly inhibited lipid accumulation in HepG2 cell at the concentration of 50μmol/L. Mechanism research demonstrated that LRTPG increased the levels of phospho-AMP-activated protein kinase and phospho-sterol regulatory element binding protein-1c in liver, further to suppress the downstream lipogenic genes as stearoyl-CoA desaturase 1, glycerol-3-phosphate acyltransferase, 1-acylglycerol-3-phosphate O-acyltransferase 2, and diacylglycerol acyltransferase 2. In addition, LRTPG increased the hydrolysis of circulating TG by up-regulating lipoprotein lipase activities. These results indicate that LRTPG prevents hyperlipidemia via activation of hepatic AMP-activated protein kinase-sterol regulatory element binding protein-1c pathway.