Full Answer
What is the HepG2 cell line?
A patent for the HepG2 cell line, “a human hepatoma-derived cell line”, was filed in 1980 by researchers at the Wistar Institute.
Can HepG2 cells predict the metabolism and elimination of xenobiotics?
Furthermore, the shortage of uptake transporters and phase I enzymes is observed in HepG2 cells, which indicates the need for careful use of this cell line to predict the metabolism and elimination of xenobiotics in hepatocytes.
Are enzymes of the UGT family associated with HepG2 cell expression?
Thus, the expression of enzymes of the UGT family—UGT1A1, UGT1A4, UGT1A6, UGT2B7, UGT2B15, and GSTM1, which play a key role in the metabolism of a wide range of anticancer agents—was observed either at very low levels in the HepG2 cell line or was not detected at all in comparison with hepatocytes [ 12, 139 ].
How does fh535 inhibit the proliferation of HepG2 cells?
Liu J., Li G., Liu D., Liu J. FH535 Inhibits the Proliferation of HepG2 Cells via Downregulation of the Wnt/β-Catenin Signaling Pathway. Mol. Med. Rep. 2014; 9 :1289–1292. doi: 10.3892/mmr.2014.1928.
Does insulin increase gene expression?
Glucocorticoids and cAMP stimulate gene transcription, while insulin represses both basal and induced gene expression. However, in contrast to PEPCK, glucose induces G6Pase gene transcription, but this is also antagonised by insulin.
How long is the gene for insulin?
The human insulin gene (INS) is a small gene of 1,425 base pairs located on chromosome 11 and is composed of 3 exons separated by two introns (29).
What activates insulin gene expression?
High blood glucose levels stimulate insulin gene expression in the β-cells of the pancreas.
How many times insulin is secreted per day?
The amount of insulin produced by a lean, healthy individual is usually between 18 and 40 U/day or 0.2–0.5 U/kg/day. Because half of this amount is secreted in the basal state while the other in response to meals, the basal insulin secretion is about 0.5–1.0 U/h.
When does insulin get released?
When we eat food, glucose is absorbed from our gut into the bloodstream, raising blood glucose levels. This rise in blood glucose causes insulin to be released from the pancreas so glucose can move inside the cells and be used.
How does insulin regulate gene expression?
Insulin controls gene transcription by modifying the binding of transcription factors on insulin-response elements or by regulating their transcriptional activities. Different insulin-signaling cascades have been characterized as mediating the insulin effect on gene transcription.
How is insulin gene regulated?
Regulation of insulin gene expression under normal circumstances is controlled chiefly by changes in glucose concentrations. Glucose coordinately recruits a highly sophisticated network of transcription factors and co-activators to the insulin promoter, and also prolongs the half-life of insulin mRNA.
Does insulin increase protein synthesis?
Insulin does not stimulate muscle protein synthesis during increased plasma branched-chain amino acids alone but still decreases whole body proteolysis in humans.
What is PPAR in hepatoma?
Here we have shown for the first time, that ligand- or insulin-mediated activation of PPARγ in human hepatoma cell line HepG2 causes the downregulation of C3 gene expression and protein secretion. PPARγ is indispensable for insulin-dependent suppression of C3, as its inactivation prevents this process from occurring. Activation of PPARγ, a nuclear receptor that plays an important role in insulin signaling, is capable of ameliorating the severe effects of insulin resistance on metabolism ( Giorgino et al., 2009; Olefsky and Glass, 2010 ). Earlier it was shown that MAP kinases are capable of phosphorylating and activating PPARγ in different cell lines ( Burns and Vanden Heuvel, 2007 ). Insulin activates MEK1/2-Erk1/2, MEK3/6-p38 and MEK4/7-JNK pathways ( Burgering et al., 1991 ), and is therefore capable of influencing PPARγ activity through these cascades ( Burns and Vanden Heuvel, 2007; Zhang et al., 1996 ). Our experiments have shown that p38 and MEK1/2 activation is necessary for insulin-dependent downregulation of C3 gene expression. While it is still possible that p38 participates in insulin-dependent inhibition of C3 gene activity by phosphorylating PPARγ, our experiments have shown that MEK1/2 acts through a different mechanism. mTORC1 is another signaling complex activated by insulin ( Scott et al., 1998 ). mTORC1 is also capable of phosphorylating and activating PPARγ ( Li et al., 2014 ). As rapamycin blocked insulin-mediated suppression of C3 gene activity, we hypothesized that mTORC1 is necessary for insulin-induced activation of PPARγ. Indeed, transfection of HepG2 cells with siRNA against PPARγ abrogated rapamycin-induced increase in C3 gene activity.
Where are human hepatoma cells from?
Human hepatoma cell line HepG2 and murine hepatoma cell line BWTG3 were obtained from the Cell Culture Bank of the Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia. HepG2 and BWTG3 cells were cultivated in DMEM containing 10% fetal calf serum (FCS) (HyClone), 5% CO2 at 37 °C. For insulin administration, HepG2 or BWTG3 cells were seeded on 24- or 96-well plates at a density of 1 × 10 4 cells/cm 2 and cultivated for 24 h. For RNA isolation and plasmid transfection cells were plated on 96-well plates, for western blotting experiments – on 24-well plates. The culture medium was replaced with a fresh one without FCS, and cells were additionally incubated for 24 h before insulin administration. After 24 h incubation with insulin, cells were harvested, and used for RNA isolation and luciferase assays. The culture medium was used for ELISA experiments. In the experiments with PI3K, NF-κB and MAP-kinase inhibitors or PPARγ ligands, HepG2 cells were treated with them 1 h before insulin administration in the following concentrations: LY294002 (10 μM), rapamycin (25 nM), QNZ (10 nM), SP600125 (10 μM), SB203580 (10 μM), U0126 (10 μM), GW1929 (10 μM). In experiments with rapamycin cells were additionally washed 1 h after rapamycin administration and medium was replaced with DMEM without FCS.
Where are C57BL/6 mice from?
Male C57BL/6 mice were purchased from Rappolovo, the nursery of Russian Academy of Sciences (St. Petersburg, Russia). Mice used in experiments reported here were maintained and handled in accordance with institutional ethics committee guidelines, and these experiments were performed in concordance with the guide for the Care and Use of Laboratory Animals ( Guide for the Care and Use of Laboratory Animals, 2011 ). The animals were maintained on a 12:12 h light–dark cycle in a low-stress environment (20 °C, 50% humidity and low noise) and provided food and water ad libitum. Mice (6–8 weeks old, weighting 18–22 g) were intraperitoneally injected with 200 μl of sterile PBS containing GW1929 (10 μg per 1 g of mouse weight) or DMSO (as a control) and 0.04 μg per 1 g of mouse weight of insulin (∼1 IU/kg) for 24 h or 30 min and then sacrificed by cervical dislocation. The livers of sacrificed mice were used for RNA isolation and ChIP assay.
Does PPAR regulate C3 gene expression?
Earlier we have shown that nuclear receptor PPARα regulates C3 gene expression in HepG2 cells by interacting with NF-κB on its promoter ( Mogilenko et al., 2013 ). Since PPARα is an important transcription factor in the insulin signaling network ( Sugden et al., 2002) and NF-κB inhibitor QNZ canceled insulin-dependent decrease in C3 gene activity ( Fig. 1 E), we asked if it is necessary for insulin-dependent downregulation of C3 gene expression. Knockdown of PPARα in HepG2 cells led to the decrease of PPARα mRNA and protein levels ( Fig. 4 A, B) and abrogated the insulin-induced downregulation of C3 gene expression ( Fig. 4 C), which suggests that PPARα participates in the insulin-dependent regulation of C3 gene expression.
Does PPAR interact with C3 promoter?
Since PPARγ is a nuclear receptor that acts by binding to gene promoters and enhancers, we asked whether it could interact with C3 gene promoter. Chromatin immunoprecipitation showed that PPARγ could interact with C3 promoter, and cell treatment with GW1929 led to the increase in PPARγ binding. Treatment of HepG2 cells with insulin resulted in a ∼25% decrease in the level of PPARγ binding to C3 promoter, while pretreatment of cells with GW1929 for an hour before insulin administration abrogated this effect ( Fig. 5 C). As a control, we measured the interaction between PPARγ and hepatic enhancer of apoA-I gene (positive control) or GAPDH genomic DNA (negative control). In agreement with the results published earlier ( Shavva et al., 2016 ), PPARγ bound apoA-I promoter, while GW1929 administration had no influence on this interaction ( Fig. 5 D). In contrast, no interaction between PPARγ and GAPDH genomic DNA was detected.
What is the gene signature of a human hepG2 cell line?
Gene expression signature of human HepG2 cell line. Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is associated with various clinico-pathological characteristics such as genetic mutations and viral infections. Therefore, numerous laboratories look out for identifying always new putative markers for the improvement ...
Is HepG2 a pure cell?
HepG2 represents a pure cell line of human liver carcinoma, often used as HCC model due to the absence of viral infection. In this study we compare gene expression profiles associated with HepG2 (as HCC model) and normal hepatocyte cells by microarray technology.