mRNA Expression In Hepatocytes Research Articles

Matcha green tea targets the gut-liver axis to alleviate obesity and metabolic disorders induced by a high-fat diet.

Obesity induced by a high-fat diet (HFD) is an increasing global health problem, leading to many metabolic syndromes. As the emerging food additive rich in tea polyphenols, theanine, caffeine, and so on, matcha green tea has gained more and more popularity for its outstanding potential in ameliorating metabolic disorders. This study investigated the composition and antioxidant activity of matcha green tea and further explored its effects on gut–liver axis homeostasis in an HFD-induced obese mouse model. Male (7–8 weeks old) C57BL/6J mice were divided into four groups with the following dietary supplementation for 8 weeks: a normal chow diet (NCD), a normal chow diet+1.0% matcha (NCM), a high-fat diet (HFD), and a high-fat diet+1.0% matcha (HFM). The results demonstrated that matcha green tea ameliorated the development of obesity, lipid accumulation, and hepatic steatosis induced by HFD. Subsequently, dietary matcha supplementation restored the alterations in fecal bile acid profile and gut microbial composition. Meanwhile, the levels of mRNA expression in hepatocytes demonstrated that matcha intervention made significant regulatory on the multiple metabolic pathways of hosts involved in glucose, lipid, and bile acid metabolism. These findings present new evidence for matcha green tea as an effective nutritional strategy to mitigate obesity and relevant metabolic disorders through the gut–liver axis.

In vitro metabolic characterization of orbitazine, a novel derivative of the PAC-1 anticancer agent.

The in vitro evaluation of new drugs is an important step in the drug development pipeline. Orbitazine is a derivative of PAC-1 that has substituted the functional group homopiperazine ring with a piperazine ring. The purpose of this study was to assess the metabolic profile of orbitazine. Metabolism was characterized in vitro by incubating liver microsomes with metabolize orbitazine or the classical metabolic enzyme substrates. High performance liquid chromatography (HPLC) and LC-MS/MS were used to identify the parent drugs and metabolites of orbitazine or metabolic enzyme substrates. There was no difference in metabolic stability or metabolites across different species. The metabolites included a debenzyl compound and several hydroxyl compounds, defined as M1(316), M2(440), M3(422), M4(422) and M5(422). We found that orbitazine was metabolized by CYP3A4, CYP2C9 and CYP2D6 in a human liver microsomes incubation system. Orbitazine had no significant inhibitory effect on CYP1A2, CYP2B6, CYP2C9, or CYP2C19 in human liver microsomes, but showed a dose-dependent inhibition of CYP2C8, CYP2D6 and CYP3A4; and there was no orbitazine-mediated induction of CYP1A2, CYP2B6, CYP3A4 or mRNA expression in hepatocytes. This in vitro data on the metabolism of orbitazine may provide valuable information to support further clinical progression as a potential therapeutic molecule.

Biliary fibrosis drives liver repopulation and phenotype transition of transplanted hepatocytes

Current research focuses on developing alternative strategies to restore decreased liver mass prior to the onset of end-stage liver disease. Cell engraftment/repopulation requires regeneration in normal liver, but we have shown that severe liver injury stimulates repopulation without partial hepatectomy (PH). We have now investigated whether a less severe injury, secondary biliary fibrosis, would drive engraftment/repopulation of ectopically transplanted mature hepatocytes. Ductular proliferation and progressive fibrosis in dipeptidyl-peptidase IV (DPPIV)(-) F344 rats was induced by common bile duct ligation (BDL). Purified DPPIV(+)/green fluorescent protein (GFP)(+) hepatocytes were infused without PH into the spleen of BDL rats and compared to rats without BDL. Within one week, transplanted hepatocytes were detected in hepatic portal areas and at the periphery of expanding portal regions. DPPIV(+)/GFP(+) repopulating cell clusters of different sizes were observed in BDL rats but not untreated normal recipients. Surprisingly, some engrafted hepatocytes formed CK-19/claudin-7 expressing epithelial cells resembling cholangiocytes within repopulating clusters. In addition, substantial numbers of hepatocytes engrafted at the intrasplenic injection site assembled into multicellular groups. These also showed biliary "transdifferentiation" in the majority of intrasplenic injection sites of rats that received BDL but not in untreated recipients. PCR array analysis showed upregulation of osteopontin (SPP1). Cell culture studies demonstrated increased Itgβ4, HNF1β, HNF6, Sox-9, and CK-19 mRNA expression in hepatocytes incubated with osteopontin, suggesting that this secreted protein promotes dedifferentiation of hepatocytes. Our studies show that biliary fibrosis stimulates liver repopulation by ectopically transplanted hepatocytes and also stimulates hepatocyte transition towards a biliary epithelial phenotype.

Antipyretic analgesic drugs have different mechanisms for regulation of the expression of inducible nitric oxide synthase in hepatocytes and macrophages

Antipyretic analgesic drugs (including non-steroidal anti-inflammatory drugs) inhibit cyclooxygenase-2 and inducible nitric oxide synthase (iNOS), resulting in decreases of the proinflammatory mediators prostaglandin E2 and nitric oxide (NO), respectively. Both mediators are regulated by nuclear factor-kappa B (NF-κB), a key transcription factor in inflammation. Few reports have compared the efficacy and potency of anti-inflammatory drugs as NO inhibitors. In our study, we examined the effects of four popular antipyretic analgesic drugs on NO production induced in hepatocytes and macrophages. Mouse RAW264.7 macrophages treated with bacterial lipopolysaccharide showed the highest efficacy with regard to NO production; aspirin, loxoprofen, ibuprofen, and acetaminophen dose-dependently suppressed NO induction. Ibuprofen showed the highest potency in suppressing the induced production of NO. In rat hepatocytes, all the drugs inhibited interleukin 1β-induced NO production and ibuprofen and loxoprofen inhibited NO induction effectively. Unexpectedly, the potency of NO suppression of each drug in hepatocytes did not always correlate with that observed in RAW264.7 cells. Microarray analyses of mRNA expression in hepatocytes revealed that the effects of the four antipyretic analgesic drugs modulated the NF-κB signaling pathway in a similar manner to the regulation of the expression of genes associated with inflammation, including the iNOS gene. However, the affected signal-transducing molecules in the NF-κB pathway were different for each drug. Therefore, antipyretic analgesic drugs may decrease NO production by modulating the NF-κB pathway in different ways, which could confer different efficacies and potencies with regard to their anti-inflammatory effects.

Antihepatotoxic Effect of Tadehaginoside, Extracted from Tadehagi triquetrum (L.), Against CCl4-Lesioned Rats Through Activating the Nrf2 Signaling Pathway and Attenuating the Inflammatory Response

Recently, an increasing number of studies suggest that oxidative stress and inflammation are associated with hepatocellular injuries. Thus, we aimed to evaluate the potential hepatoprotective role of tadehaginoside (TA) on liver lesions induced by carbon tetrachloride (CCl4). The results in vitro suggested that TA dose-dependently suppressed the cell proliferation of HepG2 cells, whereas the phosphorylated level of IκBα in cells was effectively inactivated. The study in vivo showed that TA significantly lowered the serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), immunoglobulin E (IgE), and leukotriene (LT) in CCl4-lesioned rats. Pathological examination indicated that CCl4-induced hepatocellular damage was effectively mitigated by TA treatment. Meanwhile, the contents of γ-glutamylcysteine synthetase (γ-GCS), glutathione (GSH), and catalase (CAT) in liver tissue were gradually elevated. In addition, cytochrome c oxidase (COX) mRNA expression in hepatocytes was markedly upregulated, and nuclear factor E2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keapl) levels were progressively increased. Furthermore, the tumor necrosis factor alpha (TNF-α) and nuclear factor-kappa B (NF-κB)-expressed protein were downregulated. These findings demonstrate that tadehaginoside effectively protects against CCl4-induced oxidative injury and inflammatory reaction in hepatocytes, in which the underlying mechanisms are involved in activating the Nrf2 signaling pathway and inhibiting the NF-κB pathway, thereby attenuating oxidative stress and reducing the inflammation in liver cells.

Analysis Of The Molecular Mechanisms Leading To Significantly Higher Serum Hepcidin and More Severe Anemia In Multicentric Castleman's Disease (MCD) Patients Than In Rheumatoid Arthritis (RA) Patients

BackgroundHepcidin is a key regulator of body iron homeostasis and its increase in synthesis is implicated in anemia of inflammation (AI), which is commonly observed in patients with chronic inflammatory disorders such as MCD and RA. Inflammatory cytokines, mainly interleukin-6 (IL-6), play a central role in hepcidin induction during inflammation. However, tumor necrosis factor-a (TNF-a) does not induce but rather inhibits hepcidin expression in vitro and in vivo. The bone morphogenetic proteins (BMP) and erythropoietin (EPO) are the known positive and negative regulators of hepcidin expression. Our preliminary data has showed that MCD patients have more severe anemia and higher serum hepcidin-25 concentration than did RA patients. To clarify the mechanisms resulting in this difference, the activated patterns of hepcidin-regulating cytokines and their associations with serum hepcidin-25 levels and severity of anemia were analyzed in MCD and RA patients. Methods42 patients with AI (14 with MCD and 28 with RA) treated with tocilizumab (an anti-IL-6 receptor antibody) were enrolled in this study. Major iron-related parameters including serum hepcidin-25, and serum levels of cytokines including IL-6, TNF-a, BMP and EPO were measured and correlations with hepcidin-25 as well as Hb were evaluated. Effects of cytokines on IL-6-induced hepcidin expression were analyzed in hepatoma cells by quantitative real-time PCR. ResultsThe mean levels of hepcidin-25 at baseline was significantly higher (44.6 ng/ml), and Hb was significantly lower (9.2 g/dL) in MCD, than those in RA (28.6 ng/ml for hepcidin-25, 11.2 g/dL for Hb). There were significant and positive correlations of serum hepcidin-25 levels with serum ferritin and CRP in both groups (r=0.67 and 0.68 for ferritin, and r=0.41 and 0.36 for CRP, p< 0.001 respectively for both). In contrast, serum hepcidin-25 levels did not show any significant correlation with the levels of serum IL-6 or BMP or TNF-a (p > 0.05, respectively for both groups). The mean values of IL-6, TNF-a, BMP2, BMP4 and hepcidin-25 at baseline were elevated in two groups as compared to healthy control. Of note, we found that MCD patients showed significantly lower serum TNF-a (mean 195 pg/ml) and higher serum BMP4 (mean 193 pg/ml) concentrations than did RA patients (TNF-a= 241 pg/ml, BMP4= 92 pg/ml), although the two patient groups showed comparably elevated values for IL-6, BMP2 and EPO (p> 0.05, respectively). Significant improvements in anemia and systemic symptoms, and reductions in serum hepcidin-25 levels were observed within 2 weeks in both groups after tocilizumab treatment. In in vitro experiments, IL-6-induced hepcidin mRNA expression in hepatocytes was completely inhibited with tocilizumab and partially with TNF-a, but enhanced by BMP4 as well as MCD patient's serum. These results suggest that the negative effect of TNF-a on the IL-6-induced hepcidin was more pronounced in the RA than in MCD, in contrast, the positive effect of BMP was stronger in MCD than in RA. In addition, the finding that IL-6-induced hepcidin in hepatocytes was enhanced only by adding MCD patients' serum but not RA patients' serum, indicating the activated pattern of serum hepcidin-regulating factors in the MCD different from it in RA. ConclusionsOur results suggest that the difference between MCD and RA in serum hepcidin-25 levels is partially due to the different activated patterns of positive and negative regulators of hepcidin expression. By the evidence that treatment with tocilizumab can reduce serum hepcidin and improve AI in patients with MCD and RA, we believe that IL-6 plays an essential role in the induction of hepcidin which leads to AI in MCD and RA, although multiple factors affect hepcidin levels. Disclosures:No relevant conflicts of interest to declare.

Farnesoid X receptor activation increases cholesteryl ester transfer protein expression in humans and transgenic mice

Cholesteryl ester transfer protein (CETP) activity results in a proatherogenic lipoprotein profile. In cholestatic conditions, farnesoid X receptor (FXR) signaling by bile acids (BA) is activated and plasma HDL cholesterol (HDL-C) levels are low. This study tested the hypothesis that FXR-mediated induction of CETP contributes to this phenotype. Patients with cholestasis and high plasma BA had lower HDL-C levels and higher plasma CETP activity and mass compared with matched controls with low plasma BA (each P < 0.01). BA feeding in APOE3*Leiden transgenic mice expressing the human CETP transgene controlled by its endogenous promoter increased cholesterol within apoB-containing lipoproteins and decreased HDL-C (each P < 0.01), while hepatic CETP mRNA expression and plasma CETP activity and mass increased (each P < 0.01). In vitro studies confirmed that FXR agonists substantially augmented CETP mRNA expression in hepatocytes and macrophages dependent on functional FXR expression (each P < 0.001). These transcriptional effects are likely mediated by an ER8 FXR response element (FXRE) in the first intron. In conclusion, using a translational approach, this study identifies CETP as novel FXR target gene. By increasing CETP expression, FXR activation leads to a proatherogenic lipoprotein profile. These results have clinical relevance, especially when considering FXR agonists as emerging treatment strategy for metabolic disease and atherosclerosis.

Chalcones from Angelica keiskei increase insulin receptor (InsR) and insulin receptor substrate‐2 (IRS‐2) mRNA expression in hepatocytes of rats with type 2 diabetes

Type 2 diabetes mellitus is a chronic metabolic disease characterized by insulin resistance, of which InsR and IRS play very important roles. Chalcones, isolated from Angelica keiskei, have been reported to possess a variety of biological properties including antidiabetes. To study the effect of chalcones from Angelica keiskei on the expression of InsR and IRS‐2 in hepatocytes, the rats with type 2 diabetes were orally given chalcones with 30, 10 and 0 mg/kg.bw per day for 4 wks, respectively. Blood glucose and serum insulin were evaluated by glucose oxidase method and radioimmunoassay, respectively. Reverse transcription polymerase chain reaction (RT‐PCR) was used to determine the mRNA expression of InsR and IRS‐2. Blood glucose and serum insulin of rats with 30 mg/kg.bw chalcones were significantly lower than the diabetes control. Their mRNA expression of InsR and IRS‐2 were significantly higher than the diabetes control. It may conclude that chalcones from Angelica keiskei may up‐regulate the expression levels of InsR and IRS‐2 and improve insulin resistance of rats with type 2 diabetes. [Financial support: The Health Department of Shandong contract#QNW001]

Hepatocyte Nuclear Factor 6 Activates the Transcription of CYP3A4 in Hepatocyte-like Cells Differentiated from Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (iPSCs) are a valuable source of hepatocytes for applications in drug metabolism studies. However, the current protocols for generating iPSC-derived hepatocyte-like cells (iPSHCs) are still very inefficient, and iPSHCs do not have sufficient hepatocyte-specific features, which include expression of a series of hepatocyte-specific genes, such as those encoding cytochrome P450 (CYP). In this study, we investigated whether introduction of human hepatocyte nuclear factor 6 (HNF6) could modulate the expression of CYP3A4 and other CYP genes in iPSHCs as well as in HepG2 cells, a fetal liver cell line (HFL cells), and in hepatocytes. CYP3A4 mRNA could be detected in iPSHCs, but the expression level was very low compared with those in HepG2 cells and hepatocytes. However, the CYP3A4 mRNA levels markedly increased on introduction of HNF6 and reached one-tenth of those in hepatocytes. We also found that HNF6 introduction increased CYP3A4 gene transcription in HFL cells and HepG2 cells, which have features similar to those of fetal hepatocyte-like cells; however, it did not affect CYP3A4 mRNA expression in hepatocytes. These results suggest that HNF6 plays an important role in the gene regulation of CYP3A4 during development from the fetal period to the postnatal period.

Up-regulation of hepcidin by interleukin-6 contributes to anemia of inflammation in multicentric Castleman's disease (MCD)

Multicentric Castleman's disease (MCD) is a rare lymphoproliferative disorder characterized by overproduction of interleukin-6 (IL-6). Anemia is a common symptom of MCD but its mechanism has been poorly understood. Hepcidin is an IL-6-induced key regulator of iron metabolism and has a major role in anemia of inflammation (AI). Our study showed that treatment with tocilizumab (an anti-IL-6 receptor antibody) resulted in long-term reductions of serum hepcidin25 in MCD patients, which was accompanied by progressive normalization of iron-related parameters. In in vitro experiments, IL-6-induced hepcidin mRNA expression in hepatocytes was completely inhibited with tocilizumab and partially with erythropoietin (EPO), but enhanced by bone morphogenetic protein (BMP) and patient's serum. Our findings and evidence published elsewhere, leads us to suggest that, although multiple factors affect hepcidin levels, IL-6 plays an essential role in the induction of hepcidin which leads to anemia in MCD. Therefore, IL-6 blockage may constitute a promising molecular targeting therapy for AI.

Insulin-Regulated Srebp-1c and Pck1 mRNA Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions

Insulin regulates the transcription of genes for hepatic glucose and lipid metabolism. We hypothesized that this action may be impaired in hepatocytes from insulin resistant animals. Primary hepatocytes from insulin sensitive Zucker lean (ZL) and insulin resistant Zucker fatty (ZF) rats in ad libitum or after an overnight fasting were isolated, cultured and treated with insulin and other compounds for analysis of gene expression using real-time PCR. The mRNA levels of one insulin-induced (Srebp-1c) and one insulin-suppressed (Pck1) genes in response to insulin, glucagon, and compactin treatments in hepatocytes from ad libitum ZL and ZF rats were analyzed. Additionally, the effects of insulin and T1317 on their levels in hepatocytes from ad libitum or fasted ZL or ZF rats were compared. The mRNA levels of Srebp-1c, Fas, and Scd1, but not that of Insr, Gck and Pck1, were higher in freshly isolated hepatocytes from ad libitum ZF than that from ZL rats. These patterns of Srebp-1c and Pck1 mRNA levels remained in primary hepatocyte cultured in vitro. Insulin's ability to regulate Srebp-1c and Pck1 expression was diminished in hepatocytes from ad libitum ZF, but not ZL rats. Glucagon or compactin suppressed Srebp-1c mRNA expression in lean, but not fatty hepatocytes. However, glucagon induced Pck1 mRNA expression similarly in hepatocytes from ad libitum ZL and ZF rats. Insulin caused the same dose-dependent increase of Akt phosphorylation in hepatocytes from ad libitum ZL and ZF rats. It synergized with T1317 to induce Srebp-1c, and suppressed Pck1 mRNA levels in hepatocytes from fasted, but not that from ad libitum ZF rats. We demonstrated that insulin was unable to regulate its downstream genes' mRNA expression in hepatocytes from ad libitum ZF rats. This impairment can be partially restored in hepatocytes from ZF rats after an overnight fasting, a phenomenon that deserves further investigation.

High Insulin Concentrations Repress Insulin Receptor Gene Expression in Calf Hepatocytes Cultured in Vitro

The major role of insulin and the insulin receptor (InsR) in the liver is to mediate glucose uptake into hepatocytes to synthesize glycogen and to maintain blood glucose homeostasis. In this study, we investigated the effects of high insulin concentrations on InsR gene expression in calf hepatocytes cultured in vitro. After the cells were cultured for 72 h, insulin was added to the culture solution at final concentrations of 0, 1, 10, 100 or 1000 nM. InsR mRNA expression was determined by semi-quantitative RT-PCR. The results showed that InsR mRNA expression in hepatocytes, adjusted for β-actin expression, decreased dose dependently with increasing insulin concentration. InsR mRNA expression was similar at 1 and 10 nM insulin, but was significantly lower than that in the control. InsR expression was similar at 100 and 1000 nM insulin, but was significantly lower than that in the control, 1 and 10 nM insulin groups. These data suggest that high concentrations of insulin significantly repress InsR mRNA expression in calf hepatocytes, and this inhibition occurs in a dose-dependent manner. Further studies are needed to investigate the mechanisms underlying these effects of insulin.

The effect of capillarisin on glycochenodeoxycholic acid-induced apoptosis and heme oxygenase-1 in rat primary hepatocytes

The accumulation of hydrophobic bile acids plays a role in the induction of apoptosis and necrosis of hepatocytes during cholestasis. Glycochenodeoxycholate acid (GCDC) triggers a rapid oxidative stress response as an event of glutathione (GSH) depletion and nuclear factor kappa B (NF-kappaB) activation. We therefore investigated whether the bioactivity of the antioxidant capillarisin (Cap) prevents GCDC-induced hepatocyte damage. Isolated rat hepatocytes were co-incubated with 100 muM GCDC and 0.5 mg/ml Cap for 4 h. GSH depletion and thiobarbituric acid-reactive substances (TBARS, measure of lipid peroxidation) increased after GCDC exposure, but were markedly suppressed by Cap treatment. Cap protected hepatocytes from a GCDC-induced increase in reactive oxygen species (ROS) generation and mitochondrial membrane potential induction, as measured by flow cytometry analysis. In addition, Cap was shown to inhibit GCDC-mediated NF-kappaB activation by using electrophoretic mobility shift assays (EMSA). In contrast to GCDC, Cap not only significantly decreased cytochrome c release and caspase-3 enzyme activity, but also suppressed heme oxygenase-1 protein and mRNA expression in hepatocytes. These results demonstrate that Cap function as an antioxidant reduced hepatocyte injury caused by hydrophobic bile acids, perhaps by preventing generation of ROS and release of cytochrome c, thereby minimizing hepatocytes apoptosis.

Evidence of Oatp and Mdr1 in cryopreserved rat hepatocytes

Transport proteins control uptake of drugs into the liver (e.g., organic anion transporting polypeptide (Oatp)) and excretion of drugs from the liver (e.g., multidrug resistance protein 1 (Mdr1)). In this study, cryopreserved rat hepatocytes were used to investigate the effect of different culture conditions (suspension, conventional culture and sandwich culture) on the uptake of [ 3H]-taurocholate ± probenecid and the efflux of [ 3H]-vinblastine ± ketoconazole; mRNA levels of Oatp1a1, Oatp1a4, Mdr1a and Mdr1b were determined using real-time reverse transcription polymerase chain reaction (RT-PCR) and protein expression of Mdr was assessed by immunocytochemistry. The uptake of [ 3H]-taurocholate was higher in cryopreserved rat hepatocytes maintained in suspension as compared to hepatocytes in culture. A significant time dependent decline in the uptake of [ 3H]-taurocholate was noticed from day 2 to day 4 in conventional and sandwich cultures. [ 3H]-taurocholate uptake was significantly reduced using the inhibitor probenecid. Oatp mRNA expression in hepatocytes in suspension was similar to that of liver, whereas much lower levels were detected in the cultures; this was in accordance with [ 3H]-taurocholate uptake results. Mdr1 activity was assessed by accumulation of the Mdr1 selective substrate, [ 3H]-vinblastine, in hepatocytes using ketoconazole as an inhibitor. The results showed Mdr1 activity in cryopreserved rat hepatocytes in conventional and sandwich cultures. A time dependent increase in Mdr1 activity was noticed from day 2 to day 4. Mdr1 activity was not found using hepatocytes in suspension. Mdr1 mRNA expression was high in cryopreserved hepatocytes from both culture systems. Immunocytochemistry showed the Mdr protein in membranes of hepatocytes in culture as well as in that of hepatocytes in liver sections. In conclusion, the present study showed that cryopreserved rat hepatocytes maintained canalicular transport activity (Mdr1) and basolateral transport activity. Hepatocytes in suspension had a higher uptake of taurocholate with a high Oatp (1a1 and 1a4) mRNA expression as compared to hepatocytes in culture. The presence of Mdr1 in both conventional and sandwich culture was confirmed at mRNA level, by protein expression as well as transport activity.

Evaluation of Human CYP1A2 and CYP3A4 mRNA Expression in Hepatocytes from Chimeric Mice with Humanized Liver

We investigated and compared the expression of human CYPs mRNA in primary cultures of cryopreserved human hepatocytes and in chimeric mice constructed by transplanting hepatocytes from the same human donors. Analysis was performed by real-time reverse-transcription polymerase chain reaction. Initial expression levels for the 12 human CYPs mRNA in chimeric mouse hepatocytes were higher than those in human hepatocytes, but a low correlation coefficient was observed (r=0.690). After 24 h of culture, the correlation remained low (r=0.699). The medium was replaced with fresh medium without human epidermal growth factor, and after 48 h of culture, expression of the 12 human CYPs mRNA were very similar in human hepatocytes and chimeric mouse hepatocytes, and a higher correlation coefficient was observed (r=0.809). After 72 h of culture, the correlation remained high (r=0.873). The ratio of human CYP1A2 mRNA to beta-actin mRNA in chimeric mouse hepatocytes decreased quickly during the first 24 h of culture, and then remained constant. Expression profiles of human CYP1A2 mRNA in chimeric mouse hepatocytes were similar to those in human hepatocytes after exposure of beta-naphthoflavone. CYP3A4 mRNA expression was increased significantly by rifampicin (Rif) exposure in human hepatocytes, whereas Rif-induced increases in CYP3A4 mRNA expression in chimeric mouse hepatocytes was seen for two of the three donors. In conclusion, we demonstrated that expression and induction of human CYPs in human hepatocytes can be reproduced in chimeric mouse hepatocytes.

Probucol Up-Regulates Paraoxonase 1 Expression in Hepatocytes of Hypercholesterolemic Rabbits

Paraoxonase 1 (PON1), an HDL-associated enzyme, has anti-oxidative property. Probucol, a hydrophobic antioxidant drug, inhibits progression of atherosclerosis and post-angioplasty restenosis. However, the mechanism by which probucol affects atherosclerosis is not completely understood. Sixteen rabbits fed with high cholesterol diet for 8 weeks were randomly divided into two groups: (1) starch group (n = 8): maintained high cholesterol diet plus starch (500 mg/kg/d) for 6 weeks; (2) probucol group (n = 8): the same cholesterol diet plus probucol (500 mg/kg/d) for 6 weeks. Control group (n = 8) was fed with normal diet for 14 weeks. The classic in-situ two-step perfusion of the liver with collagenase IV was used to isolate the parenchymal hepatocytes. The total activity of superoxide dismutase (SOD) and malondialdehyde (MDA) and PON1 concentrations in serum were measured after 0, 8, and 14 weeks of feeding. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of PON1. Compared with control group, rabbits fed with high cholesterol diet showed higher levels of serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), all of which were significantly reduced by probucol. Probucol significantly decreased the MDA concentration but was ineffective on SOD activity. High cholesterol diet decreased serum PON1 concentration and down-regulated PON1 mRNA expression in hepatocytes. Probucol significantly increased serum PON1 level and up-regulated the mRNA expression of PON1 as compared with starch group (0.65 +/- 0.06 versus 0.46 +/- 0.05, P = 0.001). The PON1 concentration is negatively associated with MDA concentration (r = -0.86, P = 0.003) but not with the level of HDL-C. In conclusion, probucol decreased MDA concentration, and increased PON1 serum level as well as mRNA expression in hepatocytes, which may help us to understand its antioxidant and anti-atherogenic effects.

The Human Apolipoprotein AV Gene Is Regulated by Peroxisome Proliferator-activated Receptor-α and Contains a Novel Farnesoid X-activated Receptor Response Element

The newly identified apolipoprotein AV (apoAV) gene is a key player in determining plasma triglyceride concentrations. Because hypertriglyceridemia is a major independent risk factor in coronary artery disease, the understanding of the regulation of the expression of this gene is of considerable importance. We presently characterize the structure, the transcription start site, and the promoter of the human apoAV gene. Since the peroxisome proliferator-activated receptor-alpha (PPARalpha) and the farnesoid X-activated receptor (FXR) have been shown to modulate the expression of genes involved in triglyceride metabolism, we evaluated the potential role of these nuclear receptors in the regulation of apoAV transcription. Bile acids and FXR induced the apoAV gene promoter activity. 5'-Deletion, mutagenesis, and gel shift analysis identified a heretofore unknown element at positions -103/-84 consisting of an inverted repeat of two consensus receptor-binding hexads separated by 8 nucleotides (IR8), which was required for the response to bile acid-activated FXR. The isolated IR8 element conferred FXR responsiveness on a heterologous promoter. On the other hand, in apoAV-expressing human hepatic Hep3B cells, transfection of PPARalpha specifically enhanced apoAV promoter activity. By deletion, site-directed mutagenesis, and binding analysis, a PPARalpha response element located 271 bp upstream of the transcription start site was identified. Finally, treatment with a specific PPARalpha activator led to a significant induction of apoAV mRNA expression in hepatocytes. The identification of apoAV as a PPARalpha target gene has major implications with respect to mechanisms whereby pharmacological PPARalpha agonists may exert their beneficial hypotriglyceridemic actions.

Down-regulation of organic anion transporter 2 mRNA expression by nitric oxide in primary cultured rat hepatocytes.

Role of nitric oxide (NO) on the expression of organic anion transporter 2 (OAT2) located in sinusoidal domain of hepatocytes has been investigated. Effect of NO generated in vivo in rat and delivered in vitro to hepatocytes was determined. Lipopolysaccharide (LPS) and spermine NONOate (SPER/NO) were selected as the NO donors for in vivo and in vitro experiments, respectively. Constitutive basal expression of rOAT2 mRNA was detected in the normal rat liver and the level of its expression was decreased by intraperitoneal administration of LPS. This LPS-induced decrement did not occur when aminoguanidine (AG), an inhibitor of iNOS, was co-administered with LPS. The expression of rOAT2 mRNA was detected in hepatocytes, but not in the nonparenchymal cells. In the primary cultured hepatocytes obtained from normal rats (normal hepatocytes), a time-dependent decline of rOAT2 mRNA expression was observed, but not in the hepatocytes obtained from rats pretreated with gadolinium chloride (GdCl3, Gd-hepatocytes), an inhibitor of Kupffer cell activation. The decline of rOAT2 mRNA expression observed in the normal hepatocytes was enhanced by LPS treatment, but not in the Gd-hepatocytes. The LPS-dependent enhancement in the decline of rOAT2 mRNA expression did not occur when the normal hepatocytes were treated with AG or actinomycin D. When the Gd-hepatocytes were treated with SPER/NO, an NO donor, the rOAT2 mRNA expression declined markedly. Combined, our results suggest that rOAT2 mRNA expression in hepatocytes is down-regulated by NO at least at the transcriptional step.

Increase of chemokine interferon-inducible protein-10 (IP-10) in the serum of patients with autoimmune liver diseases and increase of its mRNA expression in hepatocytes.

To clarify the role of IP-10 in autoimmune liver diseases, we studied the serum levels of IP-10 in 14 patients with autoimmune hepatitis (AIH), 23 patients with primary biliary cirrhosis (PBC), and 65 patients with chronic viral hepatitis (20 type B and 45 type C). The hepatic expression of IP-10 mRNA and the correlation between the serum levels of IP-10 and clinical parameters were also evaluated. In addition to 20 healthy controls, 16 rheumatoid arthritis (RA) patients were included as an extrahepatic inflammatory disease. The serum level of IP-10 was significantly (P < 0.02) higher in patients with AIH, PBC, and chronic hepatitis B and C than in healthy controls, and it was significantly correlated (P < 0.05) with the serum levels of aspartate aminotransferase and alanine aminotransferase in patients with AIH, PBC, and chronic hepatitis B and C. The serum level of IP-10 was not elevated in RA patients. After successful treatment of AIH and chronic hepatitis C, the serum level of IP-10 decreased to the same level as in healthy volunteers. As we previously showed in cases with chronic hepatitis B or C, in situ hybridization in both AIH and PBC cases demonstrated the expression of IP-10 mRNA in hepatocytes around focal or lobular necrosis surrounded by infiltrating mononuclear cells, whereas IP-10 mRNA was not expressed in areas around the damaged bile ducts in PBC cases. The present results suggest that IP-10 is specifically produced by hepatocytes in inflammatory areas irrespective of the aetiology of hepatitis, and that IP-10 may help to recruit T cells to the hepatic lesions in autoimmune liver diseases as well as in chronic viral hepatitis.

PRL, Placental Lactogen, and GH Induce Na+/Taurocholate-Cotransporting Polypeptide Gene Expression by Activating Signal Transducer and Activator of Transcription-5 in Liver Cells

We investigated the transcriptional regulation of the Na+/taurocholate cotransporting polypeptide gene by PRL, placental lactogen, and GH. In primary hepatocytes, ovine PRL induced a dose-dependent phosphorylation and nuclear translocation of signal transducers and activators of transcription-5a and -5b, but not -1 or -3, whereas mouse placental lactogen I and rat GH activated -5a, -5b, and -1. In EMSAs, ovine PRL, mouse placental lactogen I, and rat GH increased the specific DNA binding of nuclear signal transducer and activator of transcription-5 to its consensus element in both transfected HepG2 cells and primary hepatocytes. PRL, placental lactogen I, and GH also increased Na+/taurocholate cotransporting polypeptide mRNA expression in hepatocytes from control and pregnant (mouse placental lactogen I) rats. Genistein, a phosphotyrosine kinase inhibitor, inhibited PRL-induced signal transducer and activator of transcription-5 activation and Na+/taurocholate-cotransporting polypeptide mRNA. In HepG2 cells transiently cotransfected with either the long form of the rat PRL receptor or rat GH receptor, signal transducer and activator of transcription-5a and a -5-responsive luciferase expression vector containing the Na+/taurocholate-cotransporting polypeptide promoter, mouse placental lactogen I, like ovine PRL, activated -5a via the long form of the rat PRL receptor; whereas rat GH activated -5a via rat GH receptor, leading to transactivation of the Na+/taurocholate-cotransporting polypeptide promoter. These data establish that PRL and placental lactogen I induce Na+/taurocholate-cotransporting polypeptide gene expression via signal transducer and activator of transcription-5 proteins in liver, and indicate that these hormones play an important role in regulating liver metabolic function.