Inducibility of carbamoylphosphate synthetase (ammonia) in cultures of embryonic hepatocytes: ontogenesis of the responsiveness to hormones

Glucocorticosteroids and cyclic AMP induce carbamoylphosphate synthetase (ammonia) (CPS) in rat hepatocytes. Using an enzyme immunoassay applied to hepatocyte cultures fixed in situ, it has been demonstrated that the capacity of hepatocytes to synthesize CPS in the presence of both hormones is present as soon as the cells become recognizable as hepatocytes. Immunochemical staining of the cultures shows that hepatocytes do not acquire or express the capacity to accumulate CPS at high rates synchronously. The average levels of CPS per hepatocyte that are observed upon hormone treatment are approx 50-fold lower in embryonic than in adult hepatocytes, corresponding with an approx 10-fold lower synthetic capacity (per gram hepatocytes) and an approx 5-fold smaller size of embryonic compared to adult hepatocytes. Carbamoylphosphate synthetase levels are therefore a good parameter in studies that aim to establish the mechanisms that underly the ontogenesis of the hepatic phenotype.     

Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal alpha-glucosidase

The levels of functional mRNA encoding glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) were examined in hepatocytes from fasted and fasted/carbohydrate-refed rats and in hepatocytes inoculated into primary culture. Functional G6PDH mRNA was assessed in a cell-free protein synthesis system in vitro. We observed that hepatocytes from fasted/carbohydrate-refed rats had a 12-fold higher level of mRNA than did hepatocytes from fasted rats. The possibility that the adrenal glucocorticoids and insulin were responsible for the increase in G6PDH mRNA in refed rats was examined by studying the effect of insulin and the synthetic glucocorticoid, dexamethasone, on the level of functional G6PDH mRNA in primary cultures of rat hepatocytes maintained in a chemically defined medium. Hepatocytes from fasted rats were inoculated into primary culture and maintained for 48 h either in the absence of hormones or in the presence of insulin alone, dexamethasone alone or both hormones together. We observed that dexamethasone alone caused a fourfold increase in G6PDH mRNA while insulin caused about a twofold increase. Both hormones together elicited an increase that was additive. A comparison of functional G6PDH mRNA levels with the effect of the hormones on G6PDH activity and relative rate of enzyme synthesis suggests that the glucocorticoid elevates the level of G6PDH mRNA within the cell without causing a concommitant increase in the rate of synthesis of the enzyme or the level of G6PDH activity. The results obtained with the primary cultures of hepatocytes indicate that insulin and the glucocorticoids are probably involved with the regulation of hepatic G6PDH mRNA. However, involvement of other hormones, such as thyroid hormone, seems likely since the induced levels of G6PDH mRNA in hepatocytes in culture was one-third of that observed in refed rats.     

Hormonal inducibility of liver-specific enzymes in cultured rat embryos

In monolayer cultures, hepatocyte-specific enzymes are inducible by hormones as soon as hepatocytes differentiate from the embryonic foregut (15-somite stage). Though offering an excellent opportunity for quantitative studies, several features of a normal cell environment are lost in such a model system. To determine the inducibility of such tissue-specific enzymes in intact organisms, rat embryos were cultured in vitro for 48 h and exposed to the hormonal factors that had been found effective in monolayer culture, viz. dexamethasone, triiodothyronine and dibutyryl cyclic AMP. Normal development of the embryos during culture in vitro was assessed by general criteria reflecting growth, morphogenesis and cytodifferentiation. Development of external features, organogenesis, the distribution of cell divisions and the appearance of tissue-specific proteins such as alpha-fetoprotein and glutamate dehydrogenase served as parameters. Despite undisturbed development of the embryos as judged by these criteria, irrespective of whether the culture was started at day 10 or at day 11 of gestation (just before, respectively after the appearance of the liver primordium), induction of hepatocyte-specific enzymes like carbamoylphosphate synthetase by hormones could not be demonstrated immunohistochemically. However, induction of this enzyme by hormones could be demonstrated in monolayers of hepatocytes isolated from such embryos after 48 h of culture, providing yet another demonstration of the adequate culture conditions. In addition, an adequate uptake of hormones by the embryo during culture could be shown with radio-actively labeled dexamethasone and triiodothyronine and with a radioreceptor assay for cyclic AMP. Therefore, the presence of factors in young embryos that inhibit tissue-specific enzyme synthesis has to be postulated.     

Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.     

The initiation of hepatocyte-specific gene expression within embryonic hepatocytes is a stochastic event

To gain insight into the mechanisms that govern the first steps of liver-specific enzyme accumulation upon hormone exposure, the initial accumulation of carbamoylphosphate synthetase, phosphoenolpyruvate carboxykinase, and arginase in monolayer cultures of Embryonic Day 14 rat hepatocytes was studied. By using different fluorescent labels the initial accumulation of two enzymes could be studied simultaneously in individual cells. Both microscopic and flow cytometric analyses showed that the initial expression of genes that are under the same hormonal control appears to lack the coordinated regulation of expression that is seen later in development. The coordination is gradually established during exposure to hormones. Once gene expression becomes coordinated, the enzyme content appears to increase continuously with time. Therefore, we postulate that within individual embryonic hepatocytes the initial intercellular heterogeneity in rate of accumulation of a particular protein may be the result of competition of different genes for an initially limiting supply of common regulatory factors, leading to random differences in the rate of accumulation of the respective gene products. This makes the initiation of liver-specific gene expression within the hepatocytes a stochastic event.     

Vasopressin and angiotensin II stimulate ureogenesis through increased mitochondrial citrulline production.

Vasopressin, angiotensin II, glucagon and epinephrine (through a cAMP-independent, alpha₁adrenergic mechanism), stimulate ureogenesis in isolated rat hepatocytes. Mitochondria, isolated from hepatocytes which were previously treated with these hormones, displayed an enhanced rate of citrulline synthesis in the presence of NH₄Cl as the nitrogen source. When mitochondria were incubated with glutamine as the nitrogen source, only those mitochondria isolated from hepatocytes previously treated with epinephrine or glucagon displayed an enhanced capacity to synthesize citrulline.When cells were incubated in the absence of extracellular calcium, the effects of vasopressin and angiotensin II on urea synthesis were abolished, whereas those of epinephrine and glucagon were only diminished. Mitochondria isolated from cells incubated under these conditions, showed that the effect of all these hormones on citrulline synthesis could still be observed. However, the effects of glucagon and epinephrine plus propranolol were larger than those of angiotensin II or vasopressin.Phosphatidylinositol labeling was significantly increased by epinephrine, vasopressin and angiotensin II both in the absence or presence of calcium. Cyclic AMP levels were significantly increased by glucagon or epinephrine but not by vasopressin or angiotensin II. The effect of epinephrine on cyclic AMP levels was blocked by propranolol both in the absence or presence of calcium.     

Effects of glucagon on biosynthesis of the mitochondrial enzyme, carbamoyl-phosphate synthase I, in primary hepatocytes and Morris hepatoma 5123D

When freshly-dispersed rat hepatocytes are maintained in primary monolayer cultures, they quickly lose their capacity to synthesize the urea cycle enzyme, carbamoyl-phosphate synthase. The ability to synthesize many other proteins, e.g., serum proteins including albumin, is retained. After an initial recovery period following cell isolation (24-48 h), glucagon is able to restore the ability of cultured hepatocytes to make carbamoyl-phosphate synthase. mRNA encoding the enzyme is about 4-times higher in hepatocytes maintained for 48 h in the presence of glucagon compared to hepatocytes without the hormone, as judged by in vitro translational assays. The level of carbamoyl-phosphate synthase activity expressed in transformed hepatocytes is unique to each hepatoma. Here we show that Morris hepatoma 5123D has retained such expression, and actively synthesizes the enzyme when 5123D cells are placed in monolayer cultures. Unlike normal hepatocytes, however, synthesis continues uninterrupted at a high level whether or not glucagon is present. 5123D has higher levels of translatable carbamoyl-phosphate synthase mRNA than normal liver.     

Accumulation of carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase mRNA in embryonic rat hepatocytes. Evidence for translational control during the initial phases of hepatocyte-specific gene expression in vitro

The aim of this study was to establish whether the initial accumulation of hepatocyte-specific proteins after hormone induction is regulated at the pretranslational and/or the translational level. To this end, mRNA molar concentrations were determined and compared with rates of protein synthesis from previous studies [van Roon, M.A., Charles, R. & Lamers, W.H. (1987) Eur. J. Biochem. 165, 229-234]. In vivo, carbamoylphosphate-synthetase mRNA starts to accumulate at day 17 of pregnancy. Phosphoenolpyruvate-carboxykinase mRNA starts to accumulate only just prior to birth. Embryonic day 14 (i.e. 8 days before the expected day of birth), livers were chosen to study the regulation of the initiation of hepatocyte-specific mRNA accumulation in vitro. Accumulation of carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase mRNA is regulated by the same hormones as accumulation of the respective proteins. The rate at which carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase mRNA molecules accumulate in cultured embryonic hepatocytes is relatively low, compared to that of postnatal hepatocytes. However, the increase of the rate of synthesis of carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase protein is even 3-6-fold slower than that of mRNA. This shows that initially mRNAs accumulate intracellularly to a relatively high concentration without being efficiently translated or translatable. Only after the mRNA concentration reaches a plateau of 72 h and 48 h respectively, the cellular capacity to synthesize the respective proteins increases. Therefore, the translational efficiency is certainly one of the major rate-limiting factors of the initial phases of expression of the hepatocyte-specific genes for carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase.     

The effect of ethanol, alone and in combination with the glucocorticoids and insulin, on glucose-6-phosphate dehydrogenase synthesis and mRNA in primary cultures of hepatocytes.

The hormonal regulation of the relative rate of synthesis and mRNA of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of adult-rat liver parenchymal cells maintained in a chemically defined medium. Maintenance of hepatocytes from starved animals in a culture medium devoid of any hormones resulted in a 4-fold increase in the relative rate of G6PDH synthesis in 48 h. Parallel cultures treated with glucocorticoids alone exhibited a rate of G6PDH synthesis comparable with that in the control cultures, whereas insulin alone caused a 6.5-fold increase in the rate of synthesis in 48 h. However, if the cultures were treated with glucocorticoids and insulin simultaneously, a 13-fold increase in the rate of synthesis was observed. The effect of ethanol, alone and in combination with the hormones, on the relative rate of G6PDH synthesis was studied also. Ethanol alone caused an 8-fold increase in the rate of synthesis in 48 h, whereas the combination of ethanol, glucocorticoid and insulin caused a 25-fold increase. The amount of functional mRNA encoding G6PDH, as measured in a cell-free translation system, was compared with enzyme activity and relative rate of enzyme synthesis. The increases in G6PDH activity and relative rate of synthesis in primary cultures of hepatocytes treated with ethanol, alone and in combination with the glucocorticoids and insulin, were paralleled by comparable increases in G6PDH mRNA. The results of this study show that the glucocorticoids acted in a permissive manner to amplify the insulin stimulation of G6PDH synthesis and that insulin, glucocorticoids and ethanol interact to stimulate synthesis of G6PDH primarily by increasing the concentration of functional G6PDH mRNA.     

Effects of dexamethasone and insulin on the acute phase response of Morris hepatoma cells and of rat hepatocytes in culture

Dexamethasone and insulin stimulate production of several plasma proteins in primary cultures of adult rat hepatocytes but inhibit their production in primary cultures of Morris hepatoma cell line 7777W. The acute phase response elicited in cultured cells by crude cytokines from activated rat peritoneal macrophages is considerably higher in hepatocytes in the presence of hormones, and especially of dexamethasone. In hepatoma cells the hormones enhance the cytokine-induced formation of fibrinogen and cysteine proteinase inhibitor but are without significant effect on suppression of albumin and alpha-fetoprotein synthesis by macrophage supernatants.     

The function of gamma-glutamyl transpeptidase as a determinant in cell sensitivity to azaserine toxicity

The enzyme gamma-glutamyl transpeptidase (GGT) is characteristically present at high levels in mammalian cells that are vulnerable in vivo to the selectively toxic and carcinogenic effects of the naturally occurring diazo amino acid L-azaserine. The possible role of GGT as a determinant of cellular sensitivity to azaserine toxicity was investigated. No correlation was found between GGT activity and the abilities of different cell lines or GGT-deficient cell strains of TuWi, a human nephroblastoma-derived line high in GGT, to accumulate azaserine. However, the thiols glutathione and cysteine were found to inhibit the toxicity of azaserine in cultures of TuWi. In addition, maleate lowered both intracellular and extracellular glutathione levels and enhanced sensitivity of TuWi cells to azaserine, while serine-borate, a potent inhibitor of GGT, increased extracellular glutathione levels and inhibited azaserine toxicity. Since extracellular glutathione accumulation, which may reflect the rate of cellular glutathione turnover, is increased in cultures of azaserine-resistant, GGT-deficient strains of TuWi, we propose that GGT enhances cellular sensitivity to azaserine primarily by increasing the rate of glutathione turnover, thus removing the glutathione from detoxification pathways.     

Induction of ornithine decarboxylase in cultured mouse L cells. I. Effects of cellular growth, hormones, and actinomycin D.

The activity of ornithine decarboxylase [EC 4.1.1.17] (ODC) in mouse L cells in the confluent state was induced within 4 hr by cyclic AMP (cAMP) or by insulin. During growth of L cells the concentration of cAMP increased first, then induction of ODC occurred and finally the cell number increased: the levels of cAMP and ODC increased only transitorily and returned to the basal levels when the cells become confluent. In growing cultures, however, the presence of cAMP reduced induction of ODC and cell growth. These results suggest that cAMP is involved in induction of ODC and that its concentration may be important for enzyme induction as well as for cell growth. Actinomycin D with or without these inducers stimulated induction of ODC in L cells, whereas cycloheximide inhibited it, suggesting that these hormones affect the translational level of ODC synthesis. The effect of actinomycin D on induction of ODC was much greater in non-growing cells than in growing cells. It was also found that the half life of ODC was 81 min in non-growing cells and 112 min in growing cells. This suggests that turnover of the enzyme is more rapid in the non-growing than in the growing state and that there may be an RNA fraction which controls its turnover and which also has a very short half life.     

The effect of dexamethasone on pyruvate kinase activity in primary cultures of hepatocytes

Pyruvate kinase activity in primary cultures of hepatocytes isolated from a normal rat was maintained at a constant level similar to that found in vivo (14.0 +/- 2.8 units per mg of DNA) for over 6 days when both dexamethasone and insulin were included in the medium. Yet the pyruvate kinase activity decreased 50% when the cells were cultured for 2 days and 4 days, respectively, in the presence of either dexamethasone or insulin alone. A brief, 10 min incubation of hepatocytes in the presence of dexamethasone was sufficient to maintain the enzyme activity of cells subsequently cultured for 4 days in the presence of insulin. The optimal dexamethasone concentration was 1 microM. Three other glucocorticoids were able to maintain the pyruvate kinase activity in cells cultured in medium containing insulin. The presence of the protein synthesis inhibitors, actinomycin D or cyclohexamide in cells cultured in the presence of dexamethasone and insulin resulted in a 25% decrease in the pyruvate kinase activity. Therefore, it is suggested that the synergistic effect of glucocorticoids and insulin to maintain pyruvate kinase activity in primary cultures of hepatocytes is dependent upon the ability of these cells to maintain protein synthesis.     

Amino acid environment determines expression of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in embryonic rat hepatocytes

Summary A completely defined medium (EHM-1), which reflects the amino acid composition of fetal rat serum and contains albumin as the sole proteinaceous compound, allows the accumulation of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in the presence of dexamethasone, dibutyryl cyclic AMP, and triiodothyronine to approximately twice the level attained in a standard culture medium (RPMI 1640) supplemented with 10% fetal bovine serum (and hormones). Using the EHM-1 medium we could show that the capacity of hepatocytes to synthesize phosphoenolpyruvate carboxykinase in the presence of hormones is manifest as soon as the cells differentiate from the embryonic foregut (embryonic Day 11). Furthermore we could show that embryonic hepatocytes can become binuclear or polyploid when cultured in the presence of thyroid hormone. These investigations were supported in part by the Dutch Foundation for Medical Research FUNGO (grant 13-50-38).     

Characterization of a polyamine transport system in murine embryonic palate mesenchymal cells

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of developing systems. Ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway, has been shown to be causally related to an increase in glycosaminoglycan synthesis in murine embryonic palatal mesenchyme cells (MEPM). In order to understand other mechanisms that exist to regulate polyamine levels in cells derived from the developing craniofacial area, the present study investigated the capacity of MEPM cells to accumulate exogenous putrescine and tests the hypothesis that polyamine transport can serve as an adaptational response of MEPM cells to a change in their ability to synthesize polyamines. Transport was initiated in confluent cultures of MEPM cells by the addition of 0.1 microCi/ml of 14C-putrescine. The rate of transport, monitored for 20-120 minutes, was found to be a time-dependent saturable process. The rate of initial transport, determined by incubating MEPM cells for 15 minutes in the presence of different concentrations (1.0-20.0 microM) of 14C-putrescine, was also found to be saturable, suggesting a carrier-mediated event. Lineweaver-Burk analysis of these data revealed an apparent Km of 5.78 microM and a Vmax of 2.63 nmol/mg protein/15 minutes. Transport measured either at 4 degrees C or in the presence of 2-4 DNP was dramatically inhibited. Thus, putrescine transport is an active process, dependent upon metabolic energy. Conditions in which 1) NaCl was iso-osmotically replaced with choline chloride or 2) the Na+-electrochemical gradient was dissipated with Na+, K+-specific ionophores resulted in a decreased rate of transport indicating that putrescine transport in these cells is Na+ dependent. Noncompetitive inhibition assays utilizing sulfhydryl reagents that blocked sulfhydryl groups inhibited putrescine transport, suggesting that sulfhydryl groups are important for putrescine uptake. Competitive inhibition assays demonstrated that while spermidine and spermine inhibited putrescine uptake, ornithine did not inhibit transport. Spermidine, spermine, and putrescine thus appear to share a common transport system that is separate from that for ornithine. Putrescine transport is subject to adaptive regulation in both exponentially growing and confluent cultures of MEPM cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Improved hepatocyte culture system for studying the regulation of glycogen synthase and the effects of diabetes

When 3–4-week-old rats (young rats) are used as a source of hepatocytes, primary culture cells express the adult, differentiated, liver-specific isoform of glycogen synthase. Synthase enzyme protein levels are relatively stable over a 3 day culture period in young but not in adult (>150 g rat) hepatocyte cultures. Corresponding synthase enzyme activity and mRNA levels decrease over time in culture in adult but not in young hepatocyte cultures. Young rat hepatocytes also have the ability to proliferate in chemically defined medium in the absence of added mitogens. A diabetes-induced increase in total synthase activity has been demonstrated by our lab and others, using cultured hepatocytes, liver homogenates, and perfused livers. In the present study, utilizing synthase-specific antibody and primary cultures of cells from young normal and alloxan diabetic rats, we found that greater total synthase activity in the diabetic cells was associated with higher levels of enzyme protein. Immuneprecipitaion of ³⁵S methionine-labeled freshly plated cells demonstrates an increase in the rate of protein synthesis in diabetic as compared with normal cells. Synthase mRNA levels are correspondingly increased in the diabetic relative to normal cells. Chronic exposure of young, normal hepatocytes to increasing levels of glucose induces a dose-dependent increase in total synthase activity, total synthase protein, and synthase message levels. By comparison, cells from diabetic animals do not respond by any of these measures to increased glucose concentrations. We conclude that this defined primary culture system represents a useful model for investigating the regulation of hepatic glycogen synthase and the defects which occur as a result of diabetes. © 1996 Wiley-Liss, Inc.     

Lysosomal enzyme activities and RNA turnover rates in growing and nongrowing WI-38 and HeLa cells

Activities of three lysosomal enzymes--acid RNase. N-acetyl-beta-D-glucosaminidase and acid phosphatase--were determined during the growth cycles of WI-38 and HeLa cells, as well as in radiation-arrested WI-38 cells. In confluent and growth-arrested cultures of WI-38 cells, the lysosomal RNase increased six- to sevenfold; glucosaminidase, four- to fivefold; and phosphatase, two- to threefold. In HeLa cells, the lysosomal enzymes also increased in confluent cultures, but less than twofold; and the RNase level increased only transiently. In both WI-38 and HeLa cells, the rate of RNA breakdown also increased as cultures approached confluency. The rate of turnover of RNA, like the level of acid RNase, was higher in WI-38 cells than in HeLa cells (4 d half-life compared to 8 d). The increase in acid RNase could be prevented by incubation of cells in NH4Cl, but the rate of turnover in the presence of NH4Cl increased just as much when cells became confluent or stopped growth. The content of acid RNase could be changed more than 10-fold without altering the rate of RNA turnover. It is suggested that the increase in enzyme level is more important for possible autophagy or increased digestion of engulfed RNA, rather than for normal RNA turnover, when growth stops.     

Hormonal regulation of lipogenic enzymes in chick embryo hepatocytes in culture. Expression of the fatty acid synthase gene is regulated at both translational and pretranslational steps

Mechanisms involved in the multihormonal regulation of fatty acid synthase have been investigated by comparing levels of its mRNA with rates of enzyme synthesis in chick embryo hepatocytes in culture. Triiodothyronine or insulin caused about a 2.5-fold increase in the relative rate of synthesis of fatty acid synthase. Together, these hormones were synergistic, stimulating enzyme synthesis by nearly 40-fold (Fischer, P.W.F., and Goodridge, A.G. (1978) Arch. Biochem. Biophys. 190, 332-344). Addition of triiodothyronine stimulated increases in mRNA levels comparable to increases in enzyme synthesis whether insulin was present or not. Thus, triiodothyronine regulates fatty acid synthase primarily by controlling the amount of its mRNA. Addition of insulin, in the presence of triiodothyronine, stimulated enzyme synthesis by 14-fold and mRNA levels by only 2-fold. In the absence of triiodothyronine, insulin had no effect on mRNA levels. Thus, insulin has a major effect on the translation of fatty acid synthase mRNA. After the addition of triiodothyronine, fatty acid synthase mRNA accumulated with sigmoidal kinetics, approaching a new steady state about 48 h after the addition of hormone. Puromycin, an inhibitor of protein synthesis, blocked the effect of triiodothyronine. We suggest that the abundances of both fatty acid synthase and malic enzyme mRNAs are regulated by a common triiodothyronine-induced peptide intermediate which has a relatively long half-life. Glucagon caused an 80% decrease in the synthesis of fatty acid synthase (Fischer, P.W.F., and Goodridge, A.G. (1978) Arch. Biochem. Biophys. 190, 332-344) and a 60% decrease in the level of fatty acid synthase mRNA. Thus, glucagon regulates fatty acid synthase by controlling the concentration of its mRNA. The synthesis of malic enzyme also was inhibited by glucagon at a pretranslational step, but the inhibition was almost complete. Thus, despite coordinated regulation of the concentrations of these enzymes during starvation and refeeding, individual hormones sometimes regulate synthesis of the two enzymes at the same step and to about the same degree and sometimes at different steps or to very different degrees.     

Pretranslational regulation of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase (GTP) synthesis by glucagon and dexamethasone in adult rat hepatocytes.

The regulation of synthesis of the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and of tyrosine aminotransferase (TAT) by glucagon and glucocorticoid hormones was studied in hepatocytes maintained in suspension culture for 7 h. Specific antibodies were used to measure relative rates of enzyme synthesis after pulse-labelling of the cells with [3H]leucine or [35S]methionine. Concomitantly, amounts of mRNA were quantified after translation in vitro in a reticulocyte lysate and specific immunoprecipitation of the proteins. Glucagon stimulated the rate of synthesis of PEPCK by 4-6-fold and that of TAT by 6-8-fold in 2h. In contrast, dexamethasone had little effect on PEPCK synthesis, whereas it increased TAT synthesis by 5-9-fold. When used in combination, the two hormones displayed additive effects on TAT synthesis, whereas the glucocorticoid hormone strongly potentiated stimulation of PEPCK synthesis by glucagon. In every instance, changes in rates of synthesis of the two enzymes were totally accounted for by increases in amounts of the corresponding functional mRNA, suggesting a pretranslational site of action for both glucagon and dexamethasone.