Regulation of protein synthesis by estradiol-17beta in cultured hepatocytes

Estradiol-17beta added to cultured chick embryo hepatocytes induced the appearance in the medium of a phosphoprotein, identified as phosvitin on the basis of: (i) its behaviour on ionic exchange columns; (ii) its SDS-acrylamide gel electrophoretic mobility; (iii) its amino acid composition. The hormone treatment was also followed by a decreased synthesis of other proteins secreted by the hepatocytes.     

Regulation of glutathione S-transferase subunits 3 and 4 in cultured rat hepatocytes

mRNA levels of glutathione S-transferase (GST) subunits 3 and 4 were measured with a specific cDNA probe in adult rat hepatocytes maintained either in conventional culture or in coculture with rat liver epithelial cells. Four media conditions were used, i.e. with or without fetal calf serum (FCS) and with nicotinamide or dimethylsulfoxide (DMSO). When FCS was present in the culture medium, GST subunit 3 and 4 mRNAs were expressed at a level close to that found in freshly isolated hepatocytes during the whole culture period both in conventional culture and in coculture. All other culture conditions resulted in an increase of GST 3 and 4 mRNA levels. After exposure to phenobarbital an increase in GST 3 and 4 mRNA levels was demonstrated in both culture systems. Comparison with previous findings on the expression of GST subunits 1, 2 and 7 in the same culture conditions indicates that the different classes of GST are regulated independently.     

Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix

Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions.     

Regulation of insulin binding and glycogenesis by insulin and dexamethasone in cultured rat hepatocytes

Regulation of insulin-binding and basal (insulin-independent) as well as insulin-stimulated glycogen synthesis from [¹⁴C]glucose, net glycogen deposition and glycogen synthase activation by insulin and dexamethasone were studied in primary cultures of adult rat hepatocytes maintained under chemically defined conditions. (1) Insulin receptor number was increased in a dose-dependent fashion by dexamethasone added to the medium between 24 and 48 h of culture and reduced by insulin, whereas ligand affinity remained unaltered. Insulin-induced down-regulation of insulin receptors was not affected by the glucocorticoid. (2) Although the changes in the sensitivity to insulin of glycogen synthesis from glucose and net glycogen deposition paralleled the modulation of the number of insulin receptors, postbinding events appear to be implicated also in the regulation of insulin-sensitivity. (3) Alterations of the responsiveness of glycogen synthesis to insulin caused by the glucocorticoid and/or insulin and by variation between individual rats were inversely related to cellular glycogen contents, suggesting that hepatocellular glycogen content participates in the regulation of insulin-responsiveness of this metabolic pathway. (4) Regulation of insulin-independent glycogenesis in response to an increase from 5 to 10 mM glucose, and of insulin-dependent glycogen synthesis were different. Since the effects of this ‘physiological’ increase in exogenous glucose were small compared to the acute action of insulin, insulin rather than portal venous glucose is considered to represent the prime stimulator of hepatic glycogen synthesis.     

Regulation of insulin binding and glycogenesis by insulin and dexamethasone in cultured rat hepatocytes

Regulation of insulin-binding and basal (insulin-independent) as well as insulin-stimulated glycogen synthesis from [14C]glucose, net glycogen deposition and glycogen synthase activation by insulin and dexamethasone were studied in primary cultures of adult rat hepatocytes maintained under chemically defined conditions. Insulin receptor number was increased in a dose-dependent fashion by dexamethasone added to the medium between 24 and 48 h of culture and reduced by insulin, whereas ligand affinity remained unaltered. Insulin-induced down-regulation of insulin receptors was not affected by the glucocorticoid. Although the changes in the sensitivity to insulin of glycogen synthesis from glucose and net glycogen deposition paralleled the modulation of the number of insulin receptors, postbinding events appear to be implicated also in the regulation of insulin-sensitivity. Alterations of the responsiveness of glycogen synthesis to insulin caused by the glucocorticoid and/or insulin and by variation between individual rats were inversely related to cellular glycogen contents, suggesting that hepatocellular glycogen content participates in the regulation of insulin-responsiveness of this metabolic pathway. Regulation of insulin-dependent glycogen synthesis were different. Since the effects of this 'physiological' increase in exogenous glucose were small compared to the acute action of insulin, insulin rather than portal venous glucose is considered to represent the prime stimulator of hepatic glycogen synthesis.     

Regulation of acute phase reaction by transforming growth factor beta in cultured murine hepatocytes.

Transforming growth factor-beta (TGF beta 1), a multipotent immunoregulatory peptide produced by human platelets, has been shown to stimulate the synthesis of fibrinogen, contrapsin, complement component C3, and alpha-1-proteinase inhibitor by murine hepatocytes cultured for 2 days in DMEM containing 1 microM insulin and dexamethasone and 0.2% BSA. In the range of 10 pg to 10 ng/ml TGF-beta 1 did not elicit any change in albumin secretion. Two main inflammatory cytokines: interleukin-6 (IL-6) and interleukin-1 (IL-1), known to stimulate two different subsets of murine acute phase plasma proteins, failed to increase contrapsin and alpha-1-proteinase inhibitor production. Epidermal growth factor (EGF) in the concentration 1 ng to 10 ng/ml effectively counteracted the stimulatory effect of TGF-beta 1 on acute phase protein production. TGF-beta 1-induced fibrinogen protein levels were associated with increased beta-fibrinogen mRNA content. TGF-beta 1 appears to be an additional physiological factor responsible for the direct stimulation of normal mouse hepatocytes to acute phase response.     

Regulation of RNA degradation in cultured rat hepatocytes: Effects of specific amino acids and insulin

The regulation of RNA degradation by specific amino acids and insulin was investigated in cultured rat hepatocytes from fed rats previously injected in vivo with [6-¹⁴C]orotic acid. The effects of three groups of amino acids were compared to those of a complete amino acid mixture. The first one consisted of the eight amino acids (leucine, proline, glutamine, histidine, phenylalanine, tyrosine, methionine, tryptophan) previously found to be particularly effective in the control of proteolysis. The two other groups were defined from our study with single additions of amino acids, one consisting of proline, asparagine, glutamine, alanine, phenylalanine, and leucine and the other including the latter group with serine, histidine, and tyrosine. The results showed that these three groups were able to strongly inhibit deprivation-induced RNA breakdown at one and ten times normal plasma concentrations but to a lower extent than the complete amino acid mixture. Six amino acids (proline, asparagine, glutamine, alanine, phenylalanine, leucine) inhibited individually RNA degradation by more than 20%. However, the deletions of proline, asparagine, glutamine, or alanine from the group of these six amino acids were not followed by a loss of inhibitory effect. On the contrary, an important loss of inhibition was observed when leucine and phenylalanine were deleted. Furthermore, only these two amino acids exhibited an additive inhibitory effect. Thus leucine and phenylalanine could be considered as important inhibitors of RNA breakdown in cultured rat hepatocytes. Finally, insulin which had no significant effect on RNA degradation in the absence of amino acids, was able to potentiate the inhibitory effect of different amino acid groups. © 1993 Wiley-Liss, Inc.     

Disparate effects of monensin and colchicine on intracellular processing of secretory proteins in cultured rat hepatocytes

We have studied the biosynthesis and intracellular processing of three major secretory proteins, albumin, alpha 1-protease inhibitor and alpha 2u-globulin, in cultured rat hepatocytes. The effect of secretion-blocking agents, monensin, a monovalent ionophore, and the microtubule-affecting agents colchicine and taxol was determined. In the control cells, alpha 1-protease inhibitor, a glycoprotein, was first synthesized as an endoglycosidase-H-sensitive form with Mr 51 000, and then processed to two endoglycosidase-H-resistant forms having Mr 51 000 and 56 000, the latter of which was secreted into the medium. Initially synthesized proalbumin was converted with chase to serum-type albumin, while no pro-type precursor was identified for alpha 2u-globulin. In the cells treated with colchicine or taxol, in which secretion was greatly inhibited, the fully processed alpha 1-protease inhibitor and albumin accumulated and were finally secreted into the medium. In the monensin-treated cells, however, most of the newly synthesized alpha 1-protease inhibitor and albumin were not processed to the final mature forms, resulting in accumulation of two 51 000-Mr forms and proalbumin, respectively. Moreover in treated cells, proalbumin and the endoglycosidase-H-resistant alpha 1-protease inhibitor were finally secreted into the medium. Such an effect was not caused by NH4Cl which also inhibited the secretion and is known to exert the similar effect as monensin on the receptor-mediated endocytosis pathway. Based on these results, the use of monensin may prove valuable for more detailed analysis of intracellular processing of various proteins.     

Regulation of bile acid synthesis in cultured rat hepatocytes: stimulation by apoE-rich high density lipoproteins

Cultured rat hepatocytes obtained by liver perfusion with collagenase in the presence of soybean trypsin inhibitor were used to examine the role of high density lipoproteins (HDL) in supplying cholesterol to the hepatocyte for bile acid synthesis. Within 6 hr of adding HDL (d 1.07-1.21 g/ml) obtained from rat serum there was a significant stimulation of bile acid synthesis and secretion that reached 2-fold after 24 hr. The stimulation by HDL occurred at normal plasma concentrations (i.e., 500 micrograms/ml) and showed further stimulation in a dose-dependent manner reaching a maximum stimulation of 2- to 2.5-fold. The stimulation of bile acid synthesis was dependent on the cholesteryl ester content of the HDL. Several lines of evidence show that the HDL is taken up by a receptor-mediated process dependent on apoE. These include: 1) at the same concentration (500 micrograms/ml) apoE-poor HDL (not retained by heparin affinity chromatography of HDL isolated from the plasma of rats fasted for 72 hr stimulated bile acid synthesis by 48%, whereas apoE-rich HDL stimulated bile acid synthesis by 110%; 2) reductive methylation totally blocked the stimulation of bile acid synthesis by HDL; 3) HDLC, which contained apoE as its major protein component, also maximally stimulated bile acid synthesis; and 4) human HDL, which contained no detectable apoE, failed to stimulate bile acid synthesis. Additional studies showed that apoE-enriched HDL and HDLC both inhibited cholesterol synthesis (determined by the incorporation of 3H2O) and caused a net accumulation of cholesteryl esters in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of basal and insulin-stimulated glycogen synthesis in cultured hepatocytes. Inverse relationship to glycogen content

Cultured rat hepatocytes were used to characterize the relationship between cellular glycogen content and the basal rate, as well as response to insulin of glycogen synthesis. Depending on the concentration of medium glucose, glycogen-depleted monolayers accumulated glycogen between 24 and 48 h of culture up to the fed in vivo level. Insulin at 100 nM stimulated glycogen deposition 20-fold at 1 mM and 1.5-fold at 50 mM glucose. The rate of further glycogen storage decreased with time and increasing glycogen content. In hepatocytes preincubated with 1-50 mM glucose during 24-48 h, short-term basal and insulin-dependent incorporation of 10 mM [14C]glucose into glycogen was inversely related to the actual cellular glycogen content. This was not due to different intracellular dilution of the label, since the specific radioactivity of UDP-glucose was similar in all groups. 125I-Insulin binding indicated that insulin receptors were also not involved in this phenomenon. An inverse relationship was also found between glycogen content and the stimulation of glycogen synthase I activity by insulin, whereas the basal activity of the enzyme was dissociated from the rate of incorporation of [14C]glucose. Basal net glycogen deposition at 10 mM glucose was also inversely related to cellular glycogen; however, no such relation was evident in the presence of insulin due to the overlapping inhibition of glycogenolysis. These studies suggest that the glycogen-mediated inhibition of the activation of glycogen synthase I is operative in the cultured hepatocyte and leads to an apparent inverse relationship between the actual glycogen content and basal as well as insulin-dependent glycogenesis.     

Regulation of bile salt export pump mRNA levels by dexamethasone and osmolarity in cultured rat hepatocytes

The major canalicular bile salt export pump (Bsep) of mammalian liver is downregulated by endotoxin. This study reports on the effects of dexamethasone and osmolarity on Bsep mRNA expression in cultured rat hepatocytes and its functional relevance in rat liver. Expression of Bsep mRNA in rat hepatocytes 24 and 48 h after isolation was dependent on the presence of dexamethasone (100 nM) in the culture medium. Bsep was functionally active at the pseudocanalicular membrane in cells cultured for 4 days in medium containing dexamethasone. Hypoosmolarity (205 mosmol/l) led to an induction of Bsep mRNA levels, whereas expression was decreased by hyperosmolarity (405 mosmol/l). Also the decay of Bsep mRNA following dexamethasone withdrawal was osmosensitive. In rat liver, dexamethasone counteracted the lipopolysaccharide (LPS)-induced down-regulation of Bsep mRNA levels after 12 hours and abolished the LPS-induced inhibition of taurocholate excretion. These results indicate that glucocorticoids are strong inducers of Bsep in liver. Furthermore, Bsep mRNA levels are osmosensitively regulated. The data suggest a longterm control of Bsep mRNA by osmolarity in addition to the short-term effects on canalicular bile acid excretion, which were reported recently.     

Sialyltransferase activities in cultured rat hepatocytes

Previous studies on the age and sex dependency of the ganglioside patterns in rat liver in vivo and the concomitant determination of the activities of some enzymes involved in these pathways revealed the prominent role of the sialylation of GM3 to GD3 in determining the flow to the mono (a)- and polysialo (b)-series, respectively. Here, the influence of hormones on the activities of GM3 and GD3 synthases in isolated hepatocytes was studied. The combination of several factors (insulin, glucagon, epidermal growth factor, glucocorticoids) was found to be necessary for maintaining in vivo activity levels of GD3- but not of GM3-synthase.     

Investigation of metabolic objectives in cultured hepatocytes

Using optimization based methods to predict fluxes in metabolic flux balance models has been a successful approach for some microorganisms, enabling construction of in silico models and even inference of some regulatory motifs. However, this success has not been translated to mammalian cells. The lack of knowledge about metabolic objectives in mammalian cells is a major obstacle that prevents utilization of various metabolic engineering tools and methods for tissue engineering and biomedical purposes. In this work, we investigate and identify possible metabolic objectives for hepatocytes cultured in vitro. To achieve this goal, we present a special data-mining procedure for identifying metabolic objective functions in mammalian cells. This multi-level optimization based algorithm enables identifying the major fluxes in the metabolic objective from MFA data in the absence of information about critical active constraints of the system. Further, once the objective is determined, active flux constraints can also be identified and analyzed. This information can be potentially used in a predictive manner to improve cell culture results or clinical metabolic outcomes. As a result of the application of this method, it was found that in vitro cultured hepatocytes maximize oxygen uptake, coupling of urea and TCA cycles, and synthesis of serine and urea. Selection of these fluxes as the metabolic objective enables accurate prediction of the flux distribution in the system given a limited amount of flux data; thus presenting a workable in silico model for cultured hepatocytes. It is observed that an overall homeostasis picture is also emergent in the findings.     

Regulation of cellular glutathione peroxidase by different forms and concentrations of selenium in primary cultured bovine hepatocytes

The expression and activity of cellular glutathione peroxidase (GPx1) are regulated by selenium (Se). Generally speaking, organic forms of Se have less toxicity and greater bioavailability compared with inorganic forms. In this study, the effects of different forms and concentrations of Se on the regulation of mRNA level and activity of GPx1 in bovine hepatocytes were evaluated, and the optimal doses of different forms of Se that supported the full expression of GPx1 were determined. Primary cultured bovine hepatocyte monolayers derived from neonatal male Holstein calves (aged 1–2 days) were incubated for 24 h with 0 (control), 0.5, 1, 1.5, 2, 3, 4 or 5 μmol/L of Se from dl-selenomethionine (Se-Met), sodium selenite (Na₂SeO₃) or Kappa-selenocarrageenan (Se-Car). Compared with controls, a significantly lower level of release of lactic dehydrogenase (LDH) was observed at 0.5–5 μmol/L of Se-Met, 0.5–1 μmol/L of Na₂SeO₃ and 0.5 μmol/L of Se-Car, but significantly higher LDH release was observed at 2–5 μmol/L of Na₂SeO₃ and 3–5 μmol/L of Se-Car, and the response occurred in a dose-dependent manner. The intracellular content of reduced glutathione in all hepatocytes treated with Se was significantly lower than that of controls. Significant increases in GPx1 mRNA were obtained in all hepatocytes treated with Se, with maximal effects at 3 μmol/L of Se-Met, 1.5 μmol/L of Na₂SeO₃ and 2 μmol/L of Se-Car, respectively. Furthermore, 3 μmol/L of Se from Se-Met resulted in peak levels of GPx1 mRNA. After reaching a maximal level, higher Se supplementation led to a reduction of GPx1 mRNA. The activity of GPx1 showed similar patterns but of lower magnitude. We conclude that (a) the regulation of mRNA level and activity of GPx1 in primary cultured bovine hepatocytes by different forms of Se varies and (b) the optimal doses of Se to support the full expression of GPx1 in bovine hepatocytes when supplied as Se-Met, Na₂SeO₃ and Se-Car are 3, 1.5 and 2 μmol/L, respectively.     

Regulation of the expression of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes by glucagon and insulin

The induction of phosphoenolpyruvate carboxykinase (PEPCK) by glucagon was studied in primary rat hepatocyte cultures by determining the time course of the sequential events, increases in the enzyme's mRNA abundance, synthesis rate, amount and activity, and by investigating the antagonistic action of insulin on the induction by glucagon. 1. The mRNA of PEPCK was induced maximally 2-3 h after addition of 10 nM glucagon, as detected by Northern-blot analysis after hybridization with a biotinylated antisense RNA of PEPCK. 2. The synthesis rate of PEPCK increased maximally 2-3 h after application of glucagon as revealed by pansorbin-linked immunoprecipitation of [35S]methionine-labelled PEPCK. 3. The enzyme amount and activity was maximally induced 4 h after glucagon application. 4. The mRNA of PEPCK was half-maximally induced by 0.1 nM and maximally by 1 nM and 10 nM glucagon. The half-maximal induction by 0.1 nM glucagon was antagonized almost totally, and the maximal induction by 1 nM glucagon partially, while the maximal induction by 10 nM glucagon remained unaffected by 10 nM insulin. The results show that in cultured rat hepatocytes physiological concentrations of glucagon stimulated the induction of PEPCK by an increase in mRNA, that the glucagon-dependent increase in mRNA and enzyme-synthesis rate occurred in parallel and preceded the increase of enzyme amount and activity by 1-1.5 h, and that physiological levels of insulin antagonized the induction by glucagon in the physiological concentration range, with glucagon being the dominant hormone.     

Regulation of cytochrome P-450c by glucocorticoids and polycyclic aromatic hydrocarbons in cultured fetal rat hepatocytes

The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the synthesis of cytochrome P-450c (the major isozyme induced by polycyclic aromatic hydrocarbons) were investigated in fetal rat hepatocytes maintained in primary monolayer culture. Treatment of hepatocytes in culture with 1,2-benzanthracene resulted in a 50-fold increase in 7-ethoxycoumarin O-deethylase activity. The level of P-450c increased in the cells in a time-dependent fashion as determined by immunoelectrophoretic analysis. The inductive effect of BA was potentiated approximately 1.6- to 2.3-fold when 1 microM dexamethasone was included in the culture medium. However, dexamethasone alone had little or no effect on the induction of P-450c. The rate of synthesis of P-450c was examined by immunoisolation of the specific isozyme from total cellular proteins radiolabeled with [35S]methionine and from the protein products formed during in vitro translation of the isolated mRNA. In addition, the amount of mRNA specific for cytochrome P-450c was determined by Northern blot analysis of RNA extracted from cultured cells. The changes in the rates of synthesis and mRNA levels were found to parallel the changes in enzyme activity. The concentration of dexamethasone required to cause a half-maximal increase in P-450c content in the presence of 1,2-benzanthracene was between 10(-8) and 10(-7) M. It is concluded that glucocorticoids act synergistically with polycyclic aromatic hydrocarbons to increase the levels of P-450c expressed in the fetal rat liver, and that this action is likely mediated by the classical type II glucocorticoid receptor.     

Regulation of protein synthesis by estradiol 17 beta, dexamethasone and insulin in primary cultured Xenopus hepatocytes

Changes in the protein synthesis of Xenopus hepatocytes caused by insulin, estradiol-17 beta (estradiol) and dexamethasone were studied by using a primary culture in serum-free medium. All of these hormones stimulated the synthesis of secretory and intracellular proteins. Dexamethasone induced or stimulated the synthesis of many proteins (though limited in number), whereas estradiol induced or stimulated relatively few proteins, including the yolk precursor protein vitellogenin. The majority of these proteins differed in molecular weight and/or isoelectric point. When hepatocytes were treated with both steroids, most of the proteins were synthesized at the rates expected from the single treatment of the respective steroids. Thus, each steroid selectively stimulated the synthesis of its specific proteins. However, exceptional proteins were observed, whose syntheses were stimulated only by double treatment. In contrast, insulin seemed to cause an overall increase in individual secretory protein synthesis.