Metformin induces an agonist-specific increase in albumin production by primary cultured rat hepatocytes.

Metformin (MET) is known to increase several biological effects of insulin (INS), but there is no information concerning its direct effects on protein synthesis. We studied the action of MET on albumin production by primary cultures of freshly isolated rat hepatocytes, alone or in combination with various agonists: INS, IGF-1, EGF, thyroxin, and dexamethasone. While having no effect alone, MET in vitro potentiates the effects of INS, IGF-1, and EGF. When this increasing effect toward INS was studied over a broad concentration range, MET appeared to improve low-acting INS levels and to intensify the maximal INS effects. In contrast, MET did not change the production of albumin stimulated by thyroxin or dexamethasone. Animals chronically pretreated with MET in vivo showed a higher yield of isolated hepatocytes, better attachment, and especially higher viability after liver perfusion and during cell culture. This may largely explain why basal albumin rates were higher than in in vitro-treated cells. The effect of MET in the presence of the agonists exhibited the same agonist-specificity as in vitro. Our data provide new insights into the pharmacology of MET by showing that hepatic protein synthesis is increased by MET and INS. From the specificity of action of MET towards INS, IGF-1, and EGF (but not thyroxin or dexamethasone), we hypothesize that this biguanide may act on intracellular pathways located between membrane receptors and sites of branching in the signaling cascades shared by these agonists.

Concomitant cellular expression of heat shock regulated genes of hepatitis B virus surface antigen and of human growth hormone by a NIH-3T3 cell line.

A plasmid carrying a DNA fragment of hepatitis B virus, coding for the pre-S2 and the entire S region of the surface antigen (HBsAg), placed under the control of the promoter of the human 70 kDa heat shock protein gene (hsp70), was introduced into Line 6, a recombinant cell line that was selected from NIH-3T3 cells previously transfected with a similar construct coding for the human growth hormone cDNA gene (chGH) and with the plasmid pEJ carrying the Ha-rasEJ activated cellular oncogene. The resulting cell line, EMS8, expressed: (1) hsp70/HBsAg and hsp70/hGH hybrid genes, (2) the human Ha-rasEJ oncogene, and (3) the neomycin resistance gene, the two last plasmid markers being used for cell selection. EMS8 cells were able to carry out post-translational modifications of the middle M and the major S envelope proteins of HBV, such as assembly and glycosylation. Accordingly, the cells synthesized and secreted both free and glycosylated M and S viral proteins, and the human growth hormone protein. In addition concomitant expression of HBsAg and hGH proteins as well as their mRNA were detected in EMS8 cells at least up to 72 hr after heat induction instead of 24 hr in the case of hGH in Line 6 cells.

Expression of heat shock-regulated human growth hormone genes containing or lacking introns by NIH-3T3 and Wish cell lines.

A plasmid containing the complete genomic DNA of the human growth hormone (ghGH) comprising four introns and driven by the human promoter of the human gene of the 70 kDa heat shock protein (hsp70) has been used to transfect mouse NIH-3T3 and human Wish cells. Selected cell lines were characterized for stable hGH secretion. Similarly in the same NIH-3T3 cells, the stable expression of the same plasmid construct, but containing the complementary DNA of the hGH gene (chGH), was compared in terms of the effect of introns on heterologous protein synthesis. Genomic hGH recombined cells synthetized, in a heat regulated fashion, matured hsp70/hGH hybrid mRNA able to drive the secretion of a 22 kDa polypeptide. Like the natural hGH, this polypeptide expressed the functional hormonal activity of prolactin on casein secretion by mammary cells. The time course of hGH secretion was prolonged in ghGH transcripts, while that of mRNA degradation appeared delayed, especially in Wish cells, as compared to chGH expression. In the human Wish cells the decay of endogenous hsp mRNA has been compared to that of recombinant hsp mRNA, demonstrating that this human hsp70/hGH hybrid mRNA was present in the cytoplasm during a longer period than the human endogenous hsp70 mRNA. In conclusion, similar levels of expression and resulting gene products were expressed from the chGH or the ghGH gene in an inducible manner.

Maintenance of liver function in long term culture of hepatocytes following in vitro or in vivo Ha-rasEJ transfection.

Collagenase isolated rat hepatocytes were transfected with liposome encapsulated pEJ (LE-pEJ), a plasmid carrying the human cellular activated Ha-rasEJ oncogene. A proliferative cell line was cloned from these cells transfected in vitro. It secreted per day 0.87 micrograms albumin and 0.32 microgram transferrin per 10(6) cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminofluorene (2-AAF) into N- and ring-hydroxylated metabolites and 2-aminofluorene at rates of 1.50, 9.73, and 1.98 nmol/mg cell protein/24 hr, respectively. Rats were i.v. injected with both LE-pEJ and LE-p17hGHneo carrying the hGH cDNA gene, and secreted hGH in the plasma which induced the synthesis of anti-hGH antibodies. A cell line was cloned from cultures of primary hepatocytes isolated from the liver of transfected rats. After 2 to 3 months in culture, this cell line secreted per day 18.9 micrograms albumin and 11.0 micrograms transferrin per 10(6) cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGH per 10(6) cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 micrograms albumin and 11.7 micrograms transferrin per 10(6) cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AAF per mg cell protein. Hence, Ha-rasEJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.

Toxic effects of 7 beta-hydroxycholesterol on rat liver primary cultures, epithelial lines and co-cultures.

The toxic effects of 7 beta-hydroxycholesterol (7 beta-OHC) on cultures and co-cultures of rat hepatocytes, rat liver epithelial cell lines, and rat liver fibroblast lines were investigated. Hepatocytes in primary culture or co-cultured with proliferative epithelial cells, were not affected by the presence of 7 beta-OHC at a concentration of 400 microM over a period of 72 hours. In contrast, proliferative cultures of liver epithelial cell lines and liver fibroblast lines were killed by 50 microM 7 beta-OHC within the first 24 hours. Established liver epithelial cells (hyperploid) were more sensitive to 7 beta-OHC than the same line at early passages (diploid). When hepatocytes and liver epithelial cells were co-cultured and treated with 100 microM 7 beta-OHC, only epithelial cells were lysed. A concentration of 50 microM 7 beta-OHC was toxic to co-cultures of liver epithelial cell and fibroblasts together. In a serum-free medium, the cytotoxic concentration of 7 beta-OHC was lower than that in the serum-supplemented medium. Thus, liver epithelial cells cultured alone or co-cultured with hepatocytes were killed at 12.5 microM and 50 microM 7 beta-OHC, respectively. Finally, cholesterol concentrations four-fold that of 7 beta-OHC antagonized the lethal effects of 7 beta-OHC in the serum-free medium.

Gas chromatographic-mass spectrometric study of deacetylation and oxidation of 2-acetylaminofluorene by rat liver epithelial cell lines upon cocarcinogen induction.

Cell line cultures from postnatal and adult rats were incubated with 5-100 mumol/l [9-14C]-2-acetylaminofluorene. On incubation of 10 mumol/l, ring-hydroxylated metabolites, expressed as nmol hydroxy-2-acetylaminofluorene (OH-2-AAF)/mg cell protein/24 h, were 9-OH- 1.28 +/- 0.37, 7-OH- 1.08 +/- 0.28 and 5-OH- 0.30 +/- 0.08, and deacetylated 2-AAF (2-AF) 1.20 +/- 0.18. For 5, 10, 50 and 100 mumol/l 2-AAF, the total production of OH-2-AAF (same units) and 2-AF (%) were, respectively, 0.86 (0%), 3.86 (35%), 17.8 (60%) and 35.03 (89%). On preincubation with phenobarbital (BP) or 3-methylcholanthrene (3-MC) and then incubation of 10 mumol/l 2-AAF, the total synthesis of OH-2-AAF increased 1.9-fold (PB) and 2.5-fold (3-MC). In addition, four other OH-2-AAF (1-OH-, 3-OH- and two unknown OH-2-AAF) were produced and glucuronidation of all metabolites was induced and amounted to 57% of the total after PB and 75% after 3-MC preincubation. Metyrapone or alpha-naphthoflavone inhibition of BP or 3-MC, respectively, markedly affected the production of free and conjugated metabolites and, almost completely, the deacetylation of 2-AAF.

A serum-free medium with or without a carrier protein supports the growth of the Y-1 mouse adrenocortical cell line and its steroidogenic function.

The development of a method for the serum-free culture of the Y-1 mouse adrenocortical tumor cell line has permitted a detailed search for factors regulating cellular growth and steroidogenesis. The serum-free medium (SFM) was made of Ham's F10 basal medium supplemented with free fatty acids adsorbed on albumin. The SFM complemented with calcium, arachidonic acid and cholesterol, i.e. SFM-S, induced cell proliferation to a density at confluency higher than that obtained with 1% serum-supplemented medium (1%-SSM) and allowed to sustain cell growth for more than six passages. When albumin was replaced by a dextran polymer (Mr = 2 x 10(6)) used as a carrier of lipids instead of albumin (which resulted in a serum-free and protein-free medium, SPFM), the cell number was 75% of that observed with the SFM-S. The addition to SPFM of beta-globulin alone or combined with insulin caused a 2- or 3-fold increase in the final cell density, respectively. The ability of the Y-1 cell line to produce steroids in response to ACTH was found to be higher in SFM-S or SPFM than in 1%-SSM. Furthermore, the addition of beta-globulin to SPFM stimulated steroid hormone biosynthesis with a marked increase in 11 beta-hydroxylated steroid production. These studies demonstrate that the use of a defined mixture of nutriments and of few growth factors permits to sustain not only the cellular proliferation of the Y-1 cell line but also its differentiated function of ACTH-induced steroidogenesis.

Gas chromatography-mass spectrometry analysis of tert.-butyldimethylsilyl derivatives of 2-acetylaminofluorene and metabolites in isolated rat hepatocytes.

A new technique for the conversion of 2-acetylaminofluorene and several ring-hydroxylated metabolites to mono- and di-tert.-butyldimethylsilyl derivatives was developed to permit their analysis by gas chromatography-mass spectrometry in order to quantify the metabolism of 2-acetylaminofluorene incubated in freshly isolated rat hepatocytes. This new gas chromatography-mass spectrometry method allowed the separation, identification and quantitation of seven known metabolites comprising five arylhydroxylated compounds, 2-aminofluorene and N-hydroxy-2-acetylaminofluorene.

[Biosynthesis of metabolites of 2-acetylaminofluorene by isolated hepatocytes of rats: study by gas chromatography-mass spectrometry]. / Biosynthèse des métabolites du 2-acétylaminofluorène par des hépatocytes isolés de rat: étude par chromatographie gazeuse-spectrométrie de masse.

A new technique for the derivatization, the separation and the quantification of 2-acetylaminofluorene (2-AAF) and its metabolites biosynthetized by freshly isolated hepatocytes was developed combining gas chromatography and mass spectrometry. Analysis of the different metabolites was carried out after their derivatization into tertbutyldimethylsilyl compounds. Freshly isolated hepatocytes metabolized 2-AAF and produced five aryl-hydroxylated compounds as well as the N-hydroxy-2AAF and the 2-aminofluorene. The metabolites were found under their free and conjugated forms.

[Culture of epithelial cell lines in chemically defined media on microcarriers in biogenerators]. / Culture de lignées de cellules épithéliales dans des milieux chimiquement définis sur microsupports en biogénérateurs.

A rat liver epithelial cell line has been propagated on microcarriers in 11 or 21 laboratory culture vessels for cell culture in suspension on microcarriers (biogenerators) with Ham F10 or DME as basal synthetic culture medium either serum-supplemented (SSM), or serum-free (SFM), or serum- and protein-free (SPFM). Without serum, the use of DME allows a cellular growth in the biogenerator at least equivalent to that obtained in culture dishes. For the cultivation on microcarriers in SFM in a biogenerator the use during the first day of culture of spent serum-free medium previously incubated (SFMI) in confluent culture dishes avoids the substratum treatment with serum. Results concerning the Vero cell line cultured in SPFM are shown.