Cultures Of Fetal Hepatocytes Research Articles

Selective elimination of tumorigenic cells from mixed culture of normal and tumorigenic cells using hybrid liposomes aimed at realizing of cell therapy

While induced pluripotent stem (iPS) cells are expected to be a cell source for regenerative medicine, they also have tumorigenic properties owing to their proliferative potential. During the manufacturing of regenerative medicine products, undifferentiated iPS cells and malignant transformed cells may be mixed in the cell culture population. Therefore, it is essential to eliminate tumorigenic cells selectively. In this study, a mixed culture of normal human fetal hepatocytes (Hc cells) and human hepatocellular carcinoma cells (HuH-7 cells) was used as a cell population model to be used as regenerative medicine products, and the selective elimination of HuH-7 cells by hybrid liposomes (HL) was analyzed. HL tended to fuse and accumulate more in HuH-7 cells due to larger fluidity of plasma membrane for HuH-7 cells than that for Hc cells. In a mixed culture of Hc and HuH-7 cells, HL selectively eliminated HuH-7 cells while allowing Hc cells to remain viable. In addition, HL treatment for the mixed culture of Hc and HuH-7 cells suppressed the tumorigenicity of HuH-7 cells. Therefore, HL selectively fused and accumulated in tumorigenic cells in a mixed cell culture of normal and tumorigenic cells, and eliminated tumorigenic cells while allowing normal cells to remain viable. The results of this study suggest the potential of HL in eliminating tumorigenic cells during the manufacturing of regenerative medicine products. Thus, HL could be expected to contribute to the development of safe regenerative medical products.

Regulation of Cytosolic Sulfotransferases in Models of Human Hepatocyte Development.

Cytosolic sulfotransferases (SULTs) are expressed during early life and therefore metabolize endogenous and xenobiotic chemicals during development. Little is currently known about the regulation of individual SULTs in the developing human liver. We characterized SULT expression in primary cultures of human fetal hepatocytes and the HepaRG model of liver cell differentiation. SULT1A1 (transcript variants 1-4), SULT1C2, SULT1C4, SULT1E1, and SULT2A1 were the most abundant transcripts in human fetal hepatocytes. In HepaRG cells, SULT1B1, SULT1C2/3/4, and SULT1E1 mRNA levels increased during the transition from proliferation to confluency and then decreased as the cells underwent further differentiation. By contrast, SULT2A1 mRNA levels increased during differentiation, whereas SULT1A1 and SULT2B1 mRNA levels remained relatively constant. The temporal patterns of SULT1C2, SULT1E1, and SULT2A1 protein content were consistent with those observed at the mRNA level. To identify regulators of SULT expression, cultured fetal hepatocytes and HepaRG cells were treated with a panel of lipid- and xenobiotic-sensing receptor activators. The following effects were observed in both fetal hepatocytes and HepaRG cells: 1) liver X receptor activator treatment increased SULT1A1 transcript variant 5 levels; 2) vitamin D receptor activator treatment increased SULT1C2 and SULT2B1 mRNA levels; and 3) farnesoid X receptor activator treatment decreased SULT2A1 expression. Activators of aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, and peroxisome proliferator-activated receptors produced additional gene-dependent effects on SULT expression in HepaRG cells. These findings suggest that SULT-regulating chemicals have the potential to modulate physiologic processes and susceptibility to xenobiotic stressors in the developing human liver.

Characterization of Liver-Specific Functions of Human Fetal Hepatocytes in Culture

This study was designed to assess liver-specific functions of human fetal liver cells proposed as a potential source for hepatocyte transplantation. Fetal liver cells were isolated from livers of different gestational ages (16-22 weeks), and the functions of cell preparations were evaluated by establishing primary cultures. We observed that 20- to 22-week-gestation fetal liver cell cultures contained a predominance of cells with hepatocytic traits that did not divide in vitro but were functionally competent. Fetal hepatocytes performed liver-specific functions at levels comparable to those of their adult counterpart. Moreover, exposure to dexamethasone in combination with oncostatin M promptly induced further maturation of the cells through the acquisition of additional functions (i.e., ability to store glycogen and uptake of indocyanine green). In some cases, particularly in cultures obtained from fetuses of earlier gestational ages (16-18 weeks gestation), cells with mature hepatocytic traits proved to be sporadic, and the primary cultures were mainly populated by clusters of proliferating cells. Consequently, the values of liver-specific functions detected in these cultures were low. We observed that a low cell density culture system rapidly prompted loss of the mature hepatocytic phenotype with downregulations of all the liver-specific functions. We found that human fetal liver cells can be cryopreserved without significant loss of viability and function and evaluated up to 1 year in storage in liquid nitrogen. They might, therefore, be suitable for cell banking and allow for the transplantation of large numbers of cells, thus improving clinical outcomes. Overall, our results indicate that fetal hepatocytes could be used as a cell source for hepatocyte transplantation. Fetal liver cells have been used so far to treat end-stage liver disease. Additional studies are needed to include these cells in cell-based therapies aimed to treat liver failure and inborn errors of metabolism.

Hepatobiliary Disposition of 17-OHPC and Taurocholate in Fetal Human Hepatocytes: A Comparison with Adult Human Hepatocytes

Little information is available in the literature regarding the expression and activity of transporters in fetal human liver or cultured cells. A synthetic progesterone structural analog, 17α-hydroxyprogesterone caproate (17-OHPC), is used in the prevention of spontaneous abortion in women with a history of recurrent miscarriage (habitual abortion). 17-OHPC has been reported to traverse the placental barrier and gain access to fetal circulation. In this study, the role of transporters in the disposition of 17-OHPC in fetal and adult human hepatocytes was examined. Progesterone metabolites have been reported to induce trans-inhibition of bile acid transporter, ABCB11. Thus, we investigated the effect of 17-OHPC or its metabolites on [(3)H]taurocholic acid transport in sandwich-cultured human fetal and adult hepatocytes. 17-OHPC was taken up rapidly into the cells and transported out partially by an active efflux process that was significantly inhibited by cold temperature, cyclosporine, verapamil, and rifampin. The active efflux mechanism was observed in both adult and fetal hepatocyte cultures. 17-OHPC produced a concentration-dependent inhibition of taurocholate efflux into canaliculi in sandwich-cultured adult and fetal human hepatocytes. However, given the high concentrations required to cause inhibition of these transport processes, no adverse effects would be anticipated from therapeutic levels of 17-OHPC. We also evaluated the expression of various hepatic transporters (ABCB1, ABCB4, SLCO1B1, SLCO1B3, SLCO2B1, ABCB11, SLC10A1, ABCC2, ABCC3, ABCC4, and ABCG2) in fetal and adult hepatocytes. With the exception of ABCB4, all transporters examined were expressed, albeit at lower mRNA levels in fetal hepatocytes compared with adults.

Use of liposome encapsulated hemoglobin as an oxygen carrier for fetal and adult rat liver cell culture

Engineering liver tissue constructs with sufficient cell mass for transplantation implies culturing large numbers of hepatocytes in a reduced volume; however, providing sufficient oxygen to dense cell cultures is still not feasible using only conventional culture medium. Liposome-encapsulated hemoglobin (LEH), an oxygen-carrying blood substitute originally designed for short-term perfusion, may be a good candidate as an oxygen carrier to cultured liver cells. In this study, we investigated the feasibility of maintaining long term hepatocyte cultures using LEH. Primary fetal and adult rat liver cells were directly exposed to LEH for 6 to 14 days in static culture or in a perfused flat plate bioreactor. The functions and viability of adult rat hepatocytes exposed to LEH were not adversely affected in static monolayer culture and were even improved in the bioreactor. However, some cytotoxicity of LEH was observed with fetal rat liver cells after 4 days of culture. LEH, though a suitable oxygen carrier for long-term culture of mature hepatocytes, is not suitable in its present form for perfusing fetal hepatocyte cultures in direct contact with the liposomes; either the LEH will have to be made less toxic or a more sophisticated bioreactor that prevents the direct contact between hepatocytes and perfusates will have to be designed if fetal cells are to be used for liver tissue engineering.

The transforming growth factor‐beta (TGF‐β) mediates acquisition of a mesenchymal stem cell‐like phenotype in human liver cells

Transforming growth factor-beta (TGF-β) mediates several and sometime opposite effects in epithelial cells, inducing growth inhibition, and apoptosis but also promoting an epithelial to mesenchymal transition (EMT) process, which enhances cell migration and invasion. TGF-β plays relevant roles in different liver pathologies; however, very few is known about its specific signaling and cellular effects in human primary hepatocytes. Here we show that TGF-β inhibits proliferation and induces pro-apoptotic genes (such as BMF or BIM) in primary cultures of human fetal hepatocytes (HFH), but also up-regulates anti-apoptotic genes, such as BCL-XL and XIAP. Inhibition of the epidermal growth factor receptor (EGFR), using gefitinib, abrogates the increase in the expression of the anti-apoptotic genes and significantly enhances cell death. Simultaneously, TGF-β is able to induce an EMT process in HFH, coincident with Snail up-regulation and a decrease in E-cadherin levels, cells showing mesenchymal proteins and reorganization of the actin cytoskeleton in stress fibers. Interestingly, these cells show loss of expression of specific hepatic genes and increased expression of stem cell markers. Chronic treatment with TGF-β allows selection of a population of mesenchymal cells with a de-differentiated phenotype, reminiscent of progenitor-like cells. Process is reversible and the mesenchymal stem-like cells re-differentiate to hepatocytes under controlled experimental conditions. In summary, we show for the first time that human hepatocytes may respond to TGF-β inducing different signals, some of them might contribute to tumor suppression (growth inhibition and apoptosis), but others should mediate liver tumor progression and invasion (EMT and acquisition of a stem-like phenotype).

The regulation of IGFs and IGFBPs by prolactin in primary culture of fetal rat hepatocytes is influenced by maternal malnutrition

During perinatal development, the regulation of IGF system appears to be growth hormone (GH) independent. By using highly purified primary fetal hepatocytes, we investigated the role of prolactin (PRL) in the regulation of IGF system and hepatocyte proliferation. We also analyzed the consequence of a maternal low-protein (LP) diet on the regulation of IGF, IGF-binding protein (IGFBP), and hepatocyte proliferation by prolactin. Pregnant Wistar rats were fed a control (C) diet (20% protein) or isocaloric (LP; 8%) diet throughout gestation. On day 21.5, fetal hepatocytes were cultured for 4 days and incubated with rat prolactin. In the C hepatocytes, PRL at 100 ng/ml decreased the abundance of IGFBP-1 and IGFBP-2 by 50 (P < 0.05) and 60% (P < 0.01), respectively. It also reduced by 70% the level of IGF-II mRNA (P < 0.01). By contrast, PRL failed to modulate IGFBP-1 and IGFBP-2 production by LP hepatocytes, and this was associated with reduced abundance of the short form of PRL receptor (P < 0.05). PRL had no effect on either the proliferation or the IGF-I production by C and LP hepatocytes, although it reduced the expression of IGF-II. These results suggest that prolactin influences hepatocyte proliferation in vitro by inhibiting IGFBP-1, IGFBP-2, and IGF-II levels, which may coincide with the decline of IGF-II observed in rodents during late gestation in vivo. On the other hand, maternal LP diet induces a resistance of fetal hepatocytes to PRL.

CHARACTERIZATION OF CYTOCHROME P450 EXPRESSION IN MURINE EMBRYONIC STEM CELL-DERIVED HEPATIC TISSUE SYSTEM

An in vitro system for liver organogenesis from murine embryonic stem (ES) cells has been recently established. This system is expected to be applied to the development of a new drug metabolism assay system that uses ES cells as a substitute for animal experiments. The objective of this study was to elucidate the drug metabolism profiles of the murine ES cell-derived hepatic tissue system compared with those of primary cultures of murine adult and fetal hepatocytes. The expression of the genes of the cytochrome P450 (P450) family, such as Cyp2a5, Cyp2b10, Cyp2c29, Cyp2d9, Cyp3a11, and Cyp7a1, was observed in the murine ES cell-derived hepatic tissue system at 16 days and 18 days after plating (A16 and A18). To investigate the activities of these P450 family enzymes in the murine ES cell-derived hepatic tissue system at A16 and A18, testosterone metabolism in this system was analyzed. Testosterone was hydroxylated to 6beta-hydroxytestosterone (6beta-OHT), 16alpha-OHT, 2alpha-OHT, and 2beta-OHT in this system, and was not hydroxylated to 15alpha-OHT, 7alpha-OHT, and 16beta-OHT. This metabolism profile was similar to that of fetal hepatocytes and different from that of adult hepatocytes. Furthermore, pretreatment with phenobarbital resulted in a 2.5- and 2.6-fold increase in the production of 6beta-OHT and 16beta-OHT. Thus, evidence for drug metabolic activities in relation to P450s has been demonstrated in this system. These results in this system would be a stepping stone of the research on the development and differentiation to adult liver.

227 DIFFERENTIATION OF HEPATOCYTE-LIKE CELLS FROM MOUSE EMBRYONIC STEM CELLS IN A MONOLAYER CULTURE SYSTEM

Embryonic stem (ES) cells can be differentiated in vitro into a variety of cell lineages. In vitro differentiation of ES cells, therefore, provides a model system for organogenesis as well as an innovative approach for regenerative medicine. However, current in vitro hepatic differentiation systems from ES cells require embryoid body formation, and such systems achieve quite low differentiation efficacy. In this study, in order to examine a system for preparation of significant numbers of hepatocytes from ES cells, mouse ES cells were directly differentiated into hepatocyte-like cells using monolayer culture conditions, and their hepatic characteristics were examined. ES cells were cultured on gelatin-coated plates for 3 days in ES cell medium containing leukemia inhibitory factor (100 units mL-1) and retinoic acid (10-8 M). After 3 days, the cells were harvested by trypsinization and cultured on gelatin-coated plates for 5 days in ES cell medium containing acidic fibroblast growth factor (aFGF; 100 ng mL-1), FGF4 (20 ng mL-1), and human growth factor (50 ng mL-1). After 5 days, the cells were passaged on type I collagen-coated plates for 2 days in ES cell medium containing Oncostatin M (10 ng mL-1), followed by additional culture in modified William&amp;apos;s E medium containing 5% FBS and dexamethasone (10-7 M) for 7 days. For characterizing the differentiated ES cells, the cells were examined for albumin production by immunocytochemistry, and the expression profiles of hepatic genes by RT-PCR. For analyzing their hepatic function, the cells were examined for glycogen production by PAS staining, and their metabolic activities of ammonia elimination and urea synthesis were measured. After 10 days of culture, almost all cells showed albumin production by immunofluoresence. RT-PCR analysis showed expression of various genes specific for hepatocytes, such as ALB, AFP, TTR, and HNF3�, but not HNF4α, and genes specific for matured hepatocytes, such as PEPCK, TAT, G6P, and Cyp7a1. After 11 days of culture, glycogen production was partially detected in the hepatocyte-like cells by PAS staining. Although ammonia elimination activity of hepatocyte-like cells was shown to be significantly lower than that of the primary culture of fetal hepatocytes after 24 and 48 h of treatment (P &amp;lt; 0.05), its metabolic activity was acquired at the level of about 40% of that of fetal hepatocytes. Hepatocyte-like cells also showed lower activity of urea synthesis than fetal hepatocytes at 6, 12, and 24 h of treatment (P &amp;lt; 0.05), but this activity increased thereafter and reached about 70% of that of fetal hepatocytes after 48 h of treatment. These results demonstrated that monolayer culture conditions for ES cell differentiation could provide an in vitro system to produce significant numbers of hepatocytes. This work was supported by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan, and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

Saturated free fatty acid, palmitic acid, induces apoptosis in fetal hepatocytes in culture

To investigate the effects of saturated free fatty acid, palmitic acid (PA), on hepatocytes, we administered PA to rat hepatocytes. We demonstrated that PA inhibited the cell growth as a dose- and time-dependent manner in rat hepatocytes. PA-induced morphological changes including swelling, membrane dissolution and formation of debris, and apoptosis with appearance of sub-G1 fraction determined by cell cycle analysis after treatment for 4 days. The level of Bcl-2 was slightly decreased, in contrast, the level of Bax elevated markedly, which resulted in a significant decrease of Bcl-2/Bax ratio after PA treatment on HepG2 cells. These findings demonstrated that PA induces cell death on hepatocytes, perhaps via mitochondria-mediated apoptosis. Furthermore, the present study indicates that PA's cell toxicity may play important roles in the transition from steatosis to steatohepatitis in human especially with obesity.

Effects of Maternal Starvation on Hepatocyte Proliferation in the Late Gestation Fetal Rat

Fetal growth retardation, a common end point for a variety of conditions affecting mother and fetus, is associated with reduced liver mass. We have performed studies to determine the mechanism for decreased liver mass in a maternal starvation model of fetal growth restriction in the rat. Pregnant dams were deprived of food for 48 h before delivery on embryonic day 19 (E19). Fetal body weight was not affected. However, fetal liver weight was reduced by approximately 15%. Immunostaining of fetal liver for proliferating cell nuclear antigen and flow cytometry on isolated fetal hepatocytes showed G1 cell cycle arrest in samples from starved dams. Based on our prior studies showing attenuated hepatic insulin signaling in the late gestation fetal rat, we tested the hypothesis that G1 arrest in our model might be due to altered nutrient signaling. Fetal plasma amino acid analyses showed no decrease in branched-chain amino acids, but arginine concentrations were decreased in fetuses of fasted mothers. Reduced arginine in E19 fetal hepatocyte culture media was associated with decreased DNA synthesis. Whereas levels of cyclins D and E were unchanged in fetal hepatocytes exposed to low arginine, cyclin E-dependent kinase activity was reduced. Low arginine also induced changes in the translational machinery, indicative of impaired signaling through the nutrient sensing kinase mammalian target of rapamycin. Our results are consistent with the hypothesis that restricted nutrient availability signals to the hepatocyte cell cycle in fetuses of fasted mothers, thereby accounting for decreased hepatocyte proliferation and liver mass.

Chimeric molecule IL-6/soluble IL-6 receptor is a potent mitogen for fetal hepatocytes.

A novel recombinant molecule, termed IL-6c and consisting of a chimera of interleukin 6 (IL-6) and its soluble receptor is extremely potent in stimulating proliferation of hematopoietic progenitors. We investigated the effect of the IL-6c on the proliferation and differentiation of E14 fetal hepatocytes. IL-6c, in a dose-dependent manner, stimulated proliferation of E14 fetal rat hepatocytes. Adult hepatocyte mitogens together with IL-6c showed no further effect on proliferation. Hematopoietic stem cells mitogens SCF and flt3 ligand (FL) were also mitogenic for fetal hepatocytes, but did not further enhance the effect of IL-6c on cell proliferation. IL-6c decreased expression of fetal markers alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase, and induced expression of adult enzyme glucose-6-phosphatase (Gluc-6-P) in E14 hepatocytes. On the other hand, IL-6c strongly reduced, in a dose-dependant manner, expression of albumin and tyrosine aminotransferase (TAT). However, when the cells were grown for 3 days with IL-6c, and IL-6c was removed for the next 5 days, expression of albumin and TAT returned to levels found in control cultures. In conclusion, IL-6c stimulated proliferation and affected gene expression in fetal hepatocytes in culture.

Perfusion culture of fetal human hepatocytes in microfluidic environments

Various types of bioreactors composed of microstructured PDMS (Polydimethylsiloxane) layers have recently been fabricated for perfusion culture of mammalian cells such as adult rat hepatocytes. As a new feature of those bioreactors, in this study, cultivation of fetal human hepatocytes (FHHs) was attempted, because they have high possibility to mature in vitro with preserving their normality, which is suitable for inplantation of liver tissue equivalents reconstituted in vitro. During the perfusion culture in the PDMS bioreactors for 1 week, cells showed good attachment, spreading and reached their confluence over the channels. In addition, their albumin production was significantly enhanced in the perfusion culture using the PDMS bioreactors up to about four times during the FHH perfusion culture when compared in dish-level static culture. Hep G2 cell cultures were also performed and have also shown under perfusion conditions an enhanced cell activity multiplied by 2 compared to static conditions. Although, the cellular activities of FHH cells are still low even compared to those of the Hep G2 cells, the conclusions of this work is encouraging toward future liver tissue engineering based on in vitro propagation and maturation of hepatocyte progenitors combined with microfabrication technologies.

Establishment, characterization, and long-term maintenance of cultures of human fetal hepatocytes

Cultured human hepatocytes have broad research and clinical applications; however, the difficulties in culturing rodent and human hepatocytes are well known. These problems include the rapid loss of the hepatocytic phenotype in primary culture and the limited replicating capacity of the cultured cells. We describe the establishment of serum-free primary cultures of human fetal hepatocytes (HFHs) that retain hepatocytic morphology and gene expression patterns for several months and maintain sufficient proliferative activity to permit subculturing for at least 2 passages. Initially, HFH cultures contained 2 main cell types that morphologically resembled large and small hepatocytes. The fetal hepatocytes expressed alpha-fetoprotein (AFP), cytokeratin (CK) 19, albumin, and other hepatic proteins. Treatment of the cultures with oncostatin M (OSM) increased cell size and enhanced cell differentiation and formation of bile canaliculi, probably through an effect on hepatocyte nuclear factor (HNF) 4alpha. Approximately 1 month after plating, multiple clusters of very small cells became apparent in the cultures. These cells had very few organelles and are referred to as blast-like cells. Flow cytometric analysis of these cells showed that they express oval cell/stem cell markers such as CD90 (Thy-1), CD34, and OV-6 but do not stain with antibodies to beta(2)-microglobulin. HFH cultures maintained for 9 to 12 months produced grossly visible organoids containing ductular structures that stained for CK18, CK19, and AFP. In conclusion, HFH cultures, which might contain a population of hepatic stem cells, constitute an excellent tool for a variety of studies with human hepatocytes, including the mechanisms of viral infection.

Spheroid formation and functional restoration of human fetal hepatocytes on poly–amino acid-coated dishes after serial proliferation

Primary human fetal hepatocytes proliferated in monolayer culture up to the 9th passage. During proliferation, the cells changed their morphology from a fibroblast-like shape after inoculation to an epithelia-like polygonal shape after they reached confluence. The proliferation was associated with the loss of ammonia detoxification capacity, which is essential for the function of bioartificial liver. The cells formed spheroids on a poly-glutamic acid- or poly-aspartic acid-coated polystyrene dish that had a negatively charged surface at neutral pH. However, the cells did not form spheroids on a poly-lysine- or poly-arginine-coated dish that had a positively charged surface, which is reportedly suitable to form spheroids for adult hepatocytes. The activity of cytochrome P450 (CYP 1A1, CYP1A2) of the cells in spheroid culture was about twice as high as that of the cells in monolayer culture. The ammonia detoxification activity of the cells was restored in spheroid culture by treatment with 2% dimethylsulfoxide. These results suggest that the conditions for human fetal hepatocytes to form spheroids are different from that for adult hepatocytes, and the use of poly-glutamic acid or poly-aspartic acid coating may improve spheroid culture of proliferative human fetal hepatocytes.

Folate cofactors regulate serine metabolism in fetal ovine hepatocytes.

The fetal liver is the primary site of fetal serine production. The regulation of this unique fetal hepatic serine production is unknown. We hypothesized that serine production would be responsive to folate cofactor supply or hormonal regulation. To test this hypothesis, we determined the effect of key folate cofactors and insulin and glucagon on serine and glycine metabolism in primary culture of fetal ovine hepatocytes. Hepatocytes were cultured in serum-free, low-folate media [5 nM 5-methyl-tetrahydrofolate (THF)] with or without 50 nM 5,10-methylene-THF (MTHF) or 5-formyl-THF (FTHF). Serine and glycine production (P) and utilization (U) were determined by stable isotope dilution with [1-(13)C]serine and [1-(13)C]glycine for 24 h. The effect of insulin (1 microM) or glucagon (1 micro M) was determined in a similar manner. Under basal conditions, serine P (43.2 +/- 5.1 micromol/mg DNA per 24 h) is greater than serine U (24.1 +/- 3.1 micromol/mg DNA per 24 h), whereas glycine U (27.3 +/- 3.0 micromol/mg DNA per 24 h) exceeds glycine P (16.7 +/- 1.9 micromol/mg DNA per 24 h). MTHF results in a significant decrease in serine U (16.0 +/- 2.7 micromol/mg DNA per 24 h; p = 0.02 versus low folate), with no change in serine P. FTHF reduces serine P (36.2 +/- 4.9 micromol/mg DNA per 24 h; p = 0.01), but does not alter serine U. There were no effects on glycine metabolism with 50 nM MTHF or FTHF. Serine P and U were inversely correlated whereas glycine P and U were directly correlated with the media concentration of MTHF or FTHF. Glucagon treatment increased serine U by 260 +/- 65% versus low folate (p = 0.0004) but did not change serine P. Insulin treatment led to parallel increases in both serine P and U. Both folate cofactor availability and hormone concentrations regulate serine metabolism in the fetal liver. We speculate that serine metabolism may be a marker of fetal hepatic folate cofactor supply.

Folate Cofactors Regulate Serine Metabolism in Fetal Ovine Hepatocytes

The fetal liver is the primary site of fetal serine production. The regulation of this unique fetal hepatic serine production is unknown. We hypothesized that serine production would be responsive to folate cofactor supply or hormonal regulation. To test this hypothesis, we determined the effect of key folate cofactors and insulin and glucagon on serine and glycine metabolism in primary culture of fetal ovine hepatocytes. Hepatocytes were cultured in serum-free, low-folate media [5 nM 5-methyl-tetrahydrofolate (THF)] with or without 50 nM 5,10-methylene-THF (MTHF) or 5-formyl-THF (FTHF). Serine and glycine production (P) and utilization (U) were determined by stable isotope dilution with [1-13C]serine and [1-13C]glycine for 24 h. The effect of insulin (1 μM) or glucagon (1 μM) was determined in a similar manner. Under basal conditions, serine P (43.2 ± 5.1 μmol/mg DNA per 24 h) is greater than serine U (24.1 ± 3.1 μmol/mg DNA per 24 h), whereas glycine U (27.3 ± 3.0 μmol/mg DNA per 24 h) exceeds glycine P (16.7 ± 1.9 μmol/mg DNA per 24 h). MTHF results in a significant decrease in serine U (16.0 ± 2.7 μmol/mg DNA per 24 h;p = 0.02 versus low folate), with no change in serine P. FTHF reduces serine P (36.2 ± 4.9 μmol/mg DNA per 24 h;p = 0.01), but does not alter serine U. There were no effects on glycine metabolism with 50 nM MTHF or FTHF. Serine P and U were inversely correlated whereas glycine P and U were directly correlated with the media concentration of MTHF or FTHF. Glucagon treatment increased serine U by 260 ± 65%versus low folate (p = 0.0004) but did not change serine P. Insulin treatment led to parallel increases in both serine P and U. Both folate cofactor availability and hormone concentrations regulate serine metabolism in the fetal liver. We speculate that serine metabolism may be a marker of fetal hepatic folate cofactor supply.

STAT3 Down-regulates the Expression of Cyclin D during Liver Development

As the expression of cyclin D1 is induced during liver regeneration and also in hepatic tumor cells, cyclin D1 is likely to play an important role in the proliferation and transformation of hepatocytes. However, the role of cyclin D1 in liver development remains unknown. Here we show that the expression of D-type cyclins including cyclin D1, D2, and D3 is down-regulated along with liver development. In addition, oncostatin M (OSM), an interleukin-6 family cytokine, down-regulated the expression of cyclin D1 and D2 in a primary culture of fetal hepatocytes in which OSM induces hepatic differentiation. Ectopic expression of receptor mutants defective in the activation of either STAT3 or SHP-2/Ras indicated that the down-regulation of D1 and D2 cyclins by OSM was mediated by STAT3 but not by SHP-2/Ras. Consistently, expression of dominant negative STAT3 but not Ras relieved OSM-induced suppression of cyclin D expression. Activation of STAT3 in fetal hepatocytes of transgenic mice expressing the STAT3-estrogen receptor fusion protein by 4-hydroxytamoxifen resulted in the suppression of cyclin D1 and D2 expression. These results indicate that STAT3 activation is necessary and sufficient for down-regulation of D1 and D2 cyclins in fetal hepatocytes. Furthermore, STAT3-C, a constitutively active form of STAT3, suppressed transcription of the cyclin D1 promoter in fetal hepatocytes, whereas it activated the transcription in hepatic tumor cells, huH7 and HepG2. Thus, STAT3-mediated down-regulation of cyclin D expression is rather specific to fetal hepatocytes that are undergoing maturation processes including a reduction of their proliferation potential.

Inhibitory effect of adefovir on viral DNA synthesis and covalently closed circular DNA formation in duck hepatitis B virus-infected hepatocytes in vivo and in vitro.

The elimination of viral covalently closed circular DNA (CCC DNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with adefovir, a new acyclic nucleoside phosphonate, the prodrug of which, adefovir dipivoxil, is in clinical evaluation, is able to suppress viral CCC DNA both in vitro and in vivo using the duck HBV (DHBV) model. First, the effect of adefovir on viral CCC DNA synthesis was examined with primary cultures of DHBV-infected fetal hepatocytes. Adefovir was administered for six consecutive days starting one day before or four days after DHBV inoculation. Dose-dependent inhibition of both virion release in culture supernatants and synthesis of intracellular viral DNA was observed. Although CCC DNA amplification was inhibited by adefovir, CCC DNA was not eliminated by antiviral treatment and the de novo formation of CCC DNA was not prevented by pretreatment of the cells. Next, preventive treatment of experimentally infected ducklings with lamivudine or adefovir revealed that both efficiently suppressed viremia and intrahepatic DNA. However, persistence of viral DNA even when detectable only by PCR was associated with a recurrence of viral replication following drug withdrawal. Taken together, our results demonstrate that adefovir is a potent inhibitor of DHBV replication that inhibits CCC DNA amplification but does not effectively prevent the formation of CCC DNA from incoming viral genomes.

Regulation of IGF-I and -II by Insulin in Primary Cultures of Fetal Rat Hepatocytes

During perinatal development, insulin and nutrients, rather than GH, regulate the IGF system. A selective primary culture of fetal rat hepatocytes has been established in our laboratory to elucidate the molecular mechanism of action of the above regulatory factors on IGF-I and -II gene expression during the late fetal period of the rat. In this model we have previously reported a regulatory role for glucose on IGF-I and -II synthesis and secretion. In the same experimental model, we now report that doses of insulin (0.1–5 μm) within the physiological range in rat fetuses during the last stages of gestation evoke an increase of IGF-I and -II mRNA abundance. Insulin regulated in a parallel manner IGF peptide secretion, and an excellent correlation was observed between IGF-I and -II mRNA and IGF-I and -II peptide levels in the conditioned media in response to the hormone. Finally, the insulin-induced rise in IGF-I and -II mRNA was not mediated by stimulation of gene transcription but by increased transcript stability. The results support the hypothesis that insulin plays a major role in IGF regulation at immature stages of development.