scholarly journals GROWTH CONTROL OF DIFFERENTIATED FETAL RAT HEPATOCYTES IN PRIMARY MONOLAYER CULTURE

1974 ◽  
Vol 62 (3) ◽  
pp. 767-779 ◽  
Author(s):  
H. L. Leffert
Keyword(s):  
Dna Synthesis ◽  
Cell Attachment ◽  
Gel Filtration ◽  
Rat Hepatocytes ◽  
Hepatocyte Proliferation ◽  
Cell Multiplication ◽  
Adult Rats ◽  
Fetal Rat ◽  
Fraction I

Dialyzed fetal bovine serum contains two distinct growth-controlling macromolecular fractions: one stimulates and the other inhibits proliferation of primary cultured differentiated fetal rat hepatocytes. Both fractions are precipitated by ammonium sulfate (50% saturation, pH 7.4, 4°C). Serum fraction I (SFI, mol wt ≥ 120,000 daltons estimated by gel filtration with Bio-gel P200) appears to contain at least two factors which function, respectively, to initiate DNA synthesis (activity pH 4–10 stable) and to increase the rate at which initiated cells traverse the cell cycle (activity pH 4 and pH 10 labile). Intraperitoneal injections of SFI into adult rats have produced detectable stimulation of hepatic but not renal DNA synthesis. Serum fraction II (SFII, mol wt 40,000–80,000 daltons) suppresses in vitro incorporation of CH3-[3H]thymidine into DNA under conditions which diminish neither cell viability nor cell attachment. Mixing experiments indicate that SFI and SFII mutually antagonize each other with respect to DNA synthesis and cell multiplication. Thus, both the relative and absolute serum levels of multiple factors control in vitro fetal hepatocyte proliferation.

scholarly journals Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. IX. Specific inhibition of DNA synthesis initiation by very low density lipoprotein and possible significance to the problem of liver regeneration.

1976 ◽  
Vol 70 (1) ◽  
pp. 20-32 ◽  
Author(s):  
H L Leffert ◽  
D B Weinstein
Keyword(s):  
Dna Synthesis ◽  
Low Density Lipoprotein ◽  
Growth Control ◽  
Density Lipoprotein ◽  
Rat Hepatocytes ◽  
Hepatocyte Proliferation ◽  
Very Low Density Lipoprotein ◽  
Low Density ◽  
Rat Serum ◽  
Fetal Rat

Rat serum very low density lipoprotein (VLDL) inhibits initiation of DNA synthesis in fetal rat hepatocyte cultures; cells engaged in synthesizing DNA resist inhibition. VLDL action is specific and apparently blocks prereplicative protein synthesis. These and other results, from studies of altered blood VLDL levels and [3H] thymidine incorporation into isolated liver nuclei in 70% hepatectomized normal and mutant hyperlipoproteinemic rats, as well as from infusion studies with a "mitogenic" hormone solution, suggest that hepatic VLDL metabolism is linked to the suppression of hepatocyte proliferation.

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

2006 ◽  
Vol 291 (4) ◽  
pp. E835-E842 ◽  
Author(s):  
Ilham El Khattabi ◽  
Claude Remacle ◽  
Brigitte Reusens
Keyword(s):  
Short Form ◽  
Rat Hepatocytes ◽  
Late Gestation ◽  
Hepatocyte Proliferation ◽  
Fetal Hepatocytes ◽  
Igf System ◽  
Fetal Rat ◽  
Igf Binding

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.

scholarly journals GROWTH CONTROL OF DIFFERENTIATED FETAL RAT HEPATOCYTES IN PRIMARY MONOLAYER CULTURE

1974 ◽  
Vol 62 (3) ◽  
pp. 792-801 ◽  
Author(s):  
H. L. Leffert
Keyword(s):  
Dna Synthesis ◽  
Environmental Control ◽  
Primary Cultures ◽  
Growth Control ◽  
Rat Hepatocytes ◽  
Defined Medium ◽  
Crystalline Insulin ◽  
Fetal Rat ◽  
Primary Monolayer

The initiation of DNA synthesis has been studied in quiescent primary cultures of fetal rat hepatocytes using defined hormones and chemically defined medium. Preparations of crystalline insulin (0.01–10 µg/ml) or 20,000-fold purified somatomedin (0.01–1 µg/ml) are stimulatory. Growth hormone (0.025 µg/ml) and hydroxycortisone (0.025 µg/ml), 3':5'-GMP! (10-5 M) fail by themselves to initiate DNA synthesis but added together with insulin, enhance the stimulatory response by 50–100%. Thyroid hormones (L-T3, L-T4, 7.5–30 ng/ml) are by themselves without effect, but when they are added to thyroid hormone-depleted serum, the reconstituted mixtures show an enhanced capacity to initiate DNA synthesis. In contrast, glucagon (0.01 µg/ml) inhibits the insulin-stimulated response by about 50% without reducing basal DNA synthesis rates. The results described here and in the accompanying two reports indicate that environmental control over the initiation of DNA synthesis is complex, and can involve the participation of many factors. The in vitro findings are consistent with the concept that liver regeneration is hormonally controlled and raise the possibility that alterations of the intrahepatic ratio of insulin to glucagon are growth regulatory.

scholarly journals GROWTH CONTROL OF DIFFERENTIATED FETAL RAT HEPATOCYTES IN PRIMARY MONOLAYER CULTURE

1974 ◽  
Vol 62 (3) ◽  
pp. 780-791 ◽  
Author(s):  
K. Koch ◽  
H. L. Leffert
Keyword(s):  
Dna Synthesis ◽  
Primary Cultures ◽  
Growth Control ◽  
Rat Hepatocytes ◽  
Solvent System ◽  
Serum Factors ◽  
Conditioning Factors ◽  
Cellular Processes ◽  
Fetal Rat ◽  
Fraction I

Serum-deficient ≤0.00003% vol/vol) conditioned medium (CM) obtained from primary cultures of fetal rat hepatocytes initiates DNA synthesis and mitosis in homologous quiescent cultures. CM similarly prepared from 3T3 fibroblast cultures is inactive. At least two conditioning factors are involved in initiating DNA synthesis. The first of these, arginine, is obligatory, synthesized by the cells, and released into the culture medium. The second, a lipid or lipid-containing material, is stable to pH extremes (pH 2, pH 10) and chromatographs with an apparent R1 ∼0.5 on silica gel thin-layer plates using hexane-ether (4: 1) as the solvent system. It is suggested that these cultured hepatocytes enter or leave the G0 or early G1 phase of the cell cycle as determined in part by their capacity to use available conditioning factor and nutrient components of the medium, in particular, arginine. Serum factors including serum fraction I (4), insulin, and possibly, lipid-like conditioning material appear to initiate DNA synthesis by controlling cellular processes involved with the enhanced utilization and synthesis of growth-limiting nutrients.

scholarly journals THE LOSS OF PHENOTYPIC TRAITS BY DIFFERENTIATED CELLS

1966 ◽  
Vol 28 (3) ◽  
pp. 473-487 ◽  
Author(s):  
Joan Abbott ◽  
Howard Holtzer
Keyword(s):  
Dna Synthesis ◽  
Chondroitin Sulfate ◽  
Collagen Synthesis ◽  
Dna Interaction ◽  
Cell Multiplication ◽  
Phenotypic Traits ◽  
Embryonic Chick ◽  
Differentiated Cells ◽  
Genetically Determined

Observations were made on the behavior of chondrocytes grown under various conditions in vitro. The chondrocytes in 10-day embryonic chick vertebrae were grown as cultures of intact vertebrae, as pellets of chondrocytes liberated from their matrix, and as monodispersed cells plated out on plasma clots. Cartilage matrix was stained metachromatically with toluidine blue. Radioautographs were made of incorporated H3-thymidine, H3-proline, and S35-sulfate to determine the extent of DNA synthesis, collagen synthesis, and chondroitin sulfate synthesis, respectively. Chondrocytes in intact vertebrae or in pellets are rounded and actively synthesizing chondroitin sulfate and collagen. There is little DNA synthesis by cells in either vertebrae or pellets. Chondrocytes grown as monodisperse cells rapidly cease synthesizing cytologically detectable chondroitin sulfate and are induced to synthesize DNA and divide. There is a change in the shape of these chondrocytes from a rounded to a more stellate condition which accompanies the shift in metabolic activity. Conversely, when the cells attain a certain cell density, they reacquire a rounded shape, cease dividing, and again synthesize chondroitin sulfate. Clusters of chondrocytes synthesize more chondroitin sulfate than isolated chondrocytes. It is concluded that most chondrocytes synthesizing chondroitin sulfate do not concurrently synthesize DNA. Interaction between associated chondrocytes is important in inducing and maintaining chondroitin sulfate synthesis in genetically determined chondrocytes. Failure of interaction between chondrocytes leads to DNA synthesis and cell multiplication.

scholarly journals Hepatocyte differentiation in vitro: initiation of tyrosine aminotransferase expression in cultured fetal rat hepatocytes.

1989 ◽  
Vol 109 (6) ◽  
pp. 3403-3410 ◽  
Author(s):  
L L Shelly ◽  
W Tynan ◽  
W Schmid ◽  
G Schütz ◽  
G C Yeoh
Keyword(s):  
Enzyme Activity ◽  
Molecular Mechanisms ◽  
Rat Hepatocytes ◽  
Tyrosine Aminotransferase ◽  
Simultaneous Increase ◽  
Mrna Levels ◽  
Fetal Hepatocytes ◽  
Fetal Rat ◽  
Rat Hepatocyte Culture

A fetal rat hepatocyte culture system has been used to study the molecular mechanisms of tyrosine aminotransferase (TAT) gene expression during development. It has previously been shown that TAT activity can be detected in 19-d, but not 15-d, gestation hepatocytes on the first day of culture (Yeoh, G. C. T., F. A. Bennett, and I. T. Oliver. 1979. Biochem. J. 180:153-160). In this study enzyme activity, synthesis, and mRNA levels were determined in hepatocytes isolated from 13-, 15-, and 19-d gestation rats maintained in culture for 1, 2, or 3 d and exposed to dexamethasone. TAT expression is barely detectable in 13-d gestation hepatocytes even after 3 d in culture. Hepatocytes isolated from 15-d gestation fetuses have undetectable levels of enzyme activity and synthesis on the first day of culture; both can be assayed by days 2 and 3. TAT mRNA levels in these hepatocytes, measured by hybridization with a specific cDNA, increase substantially during culture. TAT activity, synthesis, and mRNA are evident on the first and subsequent days of culture in 19-d gestation hepatocytes. Transcription measurements in isolated nuclei indicate that the increase in TAT mRNA in 15- and 19-d gestation hepatocytes is associated with an increase in transcription of the gene. Immunocytochemical studies demonstrated that the increase in TAT expression correlated with an increase in the proportion of hepatocytes expressing the enzyme, rather than a simultaneous increase in all hepatocytes. These results support the proposal that a subpopulation of 15-d fetal hepatocytes undergo differentiation in culture with respect to TAT.

scholarly journals Transforming growth factor-α-induced DNA synthesis and c-myc expression in primary rat hepatocyte cultures is modulated by indomethacin

1992 ◽  
Vol 281 (3) ◽  
pp. 729-733 ◽  
Author(s):  
G G Skouteris ◽  
M McMenamin
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Phospholipase A2 ◽  
Culture Medium ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Receptor Gene ◽  
Hepatocyte Proliferation ◽  
Transforming Growth Factor Α

Primary hepatocytes stimulated with epidermal growth factor (EGF) secrete prostaglandins into the culture medium as soon as 1 h after the addition of the EGF. Transforming growth factor-alpha (TGF alpha), a potent hepatocyte mitogen, shares the same receptor with EGF, and its expression is increased after partial hepatectomy. TGF alpha is also secreted in culture. We have observed that TGF alpha induced hepatocyte DNA synthesis (30 h after addition) and at the same time stimulated the production of prostaglandins E2 and F2 alpha by the cultured hepatocytes. Indomethacin at 20-100 microM inhibited the TGF alpha-induced hepatocyte DNA synthesis, and this effect was specifically due to the inhibition of prostaglandin formation. Indomethacin also inhibited a TGF-alpha-induced increase in hepatocyte c-myc expression, indicating that prostaglandins mediate this increase, as previously shown for EGF. TGF alpha increased the expression of the EGF receptor gene, and this was prevented by the presence of an antibody against TGF alpha in the culture medium. We therefore suggest that TGF alpha induces hepatocyte proliferation either through coupling with its receptor (i.e. the EGF receptor) or by subsequent phosphorylation of lipocortin I. This leads to activation of phospholipase. A2, which seems to regulate the metabolism of arachidonic acid and the formation of prostaglandins. Thus hepatocyte proliferation in vitro appears to be controlled by a self-regulatory autocrine pathway involving activation of phospholipase A2 and secretion of prostaglandins and TGF alpha.

The emergence of ErbB2 expression in cultured rat hepatocytes correlates with enhanced and diversified EGF-mediated signaling

2006 ◽  
Vol 291 (1) ◽  
pp. G16-G25 ◽  
Author(s):  
Lawrence A. Scheving ◽  
Linda Zhang ◽  
Mary C. Stevenson ◽  
Eun Soo Kwak ◽  
William E. Russell
Keyword(s):  
Dna Synthesis ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Immunological Methods ◽  
Erbb Receptors ◽  
Erbb2 Expression ◽  
Egfr Expression

The proliferative effects of EGF in liver have been extensively investigated in cultured hepatocytes. We studied the effects of EGF, insulin, and other growth regulators on the expression, interaction, and signaling of ErbB receptors in primary cultures of adult rat hepatocytes. Using immunological methods and ErbB tyrosine kinase inhibitors, we analyzed the expression and signaling patterns of the ErbB kinases over 120 h of culture. Basal and EGF-stimulated protein tyrosine phosphorylation increased as cells adapted in vitro. EGF receptor (EGFr) expression declined in the first 24 h, whereas ErbB3 expression rose. Although ErbB2 was not present in freshly isolated hepatocytes, EGF and insulin independently induced ErbB2 while suppressing ErbB3 expression. Low concentrations of EGF and insulin synergistically stimulated ErbB2 expression and DNA synthesis. The greatest increase in ErbB2, which is normally expressed by fetal and neonatal hepatocytes, occurred shortly before the onset of DNA synthesis (>40 h). EGF promoted EGFr and ErbB2 coassociation, stimulating tyrosine phosphorylation of both proteins. In contrast, heregulin β1 (HRG-β1) did not promote ErbB2 and ErbB3 coassociation. A selective tyrphostin inhibitor of ErbB2 suppressed EGF-stimulated DNA synthesis, but maximum suppression required the blockade of the EGFr kinase as well. Maximal EGF stimulation of DNA synthesis in vitro depends on the induction of ErbB2 and involves an EGFr-ErbB2 heterodimer. The ability of insulin to induce ErbB2 suggests both a mechanism for the synergy between insulin and EGF and a possible metabolic control of ErbB2 in vivo.

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