The long-term effect of glucagon on pyruvate kinase activity in primary cultures of hepatocytes

Glucagon caused a marked decrease in the total L-pyruvate kinase activity of control hepatocytes maintained in monolayer culture (t1/2 = 54 h), while the addition of insulin to hepatocytes isolated from a fasted rat caused a four- to fivefold increase in the total enzyme activity. Maintenance of L-pyruvate kinase in control cultures of hepatocytes was shown to require insulin. However, when 1 microM glucagon was present in the medium, the total L-pyruvate kinase activity was not maintained even in the presence of 1 microM insulin, but rather the total L-pyruvate kinase activity of the cells steadily declined from 12.1 to 5.7 units/mg DNA by the 6th day in culture. The increase in the total L-pyruvate kinase activity of fasted hepatocytes cultured in the presence of insulin was shown to result from an increase in protein synthesis, since actinomycin D and cycloheximide blocked the insulin-induced increase in the enzyme activity. The addition of 1 microM glucagon to cultures of fasted hepatocytes also blocked the insulin-induced increase in total L-pyruvate kinase activity. Since glucagon decreased the total L-pyruvate kinase activity in control hepatocytes and blocked the increase in L-pyruvate kinase activity in fasted hepatocytes, it is suggested that, in addition to the phosphorylation of L-pyruvate kinase by a cAMP-dependent protein kinase, glucagon also acts to decrease the synthesis of L-pyruvate kinase in vitro.     

Increased messenger RNA concentration for carbamoyl-phosphate synthase II in hepatoma 3924A

Previous investigations demonstrated that carbamoyl-phosphate synthase II (synthase II) (EC 6.3.5.5) activity, amount, and in vivo synthetic rate increased approximately 9-fold in the rapidly proliferating rat hepatoma 3924A compared to normal liver. This study provides evidence by Northern and RNA dot blot hybridizations of a 13-fold increase in the amount of hepatoma 3924A synthase II mRNA compared to levels in normal liver. Southern and DNA dot blots indicated amplification of CAD hepatoma 3924A synthase II gene product.     

Rapid degradation of newly synthesized collagen by primary cultures of adult rat hepatocytes

Greater than 95% of the total radioactive hydroxyproline synthesized by adult rat hepatocytes during the first 24 to 48 hours in monolayer culture is soluble in 5% TCA. During the next 24-hour culture period, the amount of TCA-soluble hydroxyproline is reduced to approximately 40%. Likewise, rat hepatoma cells (HTC) also produce a significant amount of TCA-soluble hydroxyproline (48%). These findings suggest that hepatocytes not only have the capacity to synthesize collagen, but also have the ability to degrade this fibrous protein.     

Hormonal regulation of carbamoyl-phosphate synthetase I synthesis in primary cultured hepatocytes and Reuber hepatoma H-35. Defective regulation in hepatoma cells

Regulation of carbamoyl-phosphate synthetase I (CPS) synthesis by various hormones was compared in primary cultured hepatocytes from adult rat and in Reuber hepatoma H-35 by pulse labeling of the cells with [35S]methionine. CPS synthesis in hepatocytes was stimulated 8-fold and 5-fold by dexamethasone and glucagon respectively. CPS synthesis in hepatocytes was synergically (about 50-fold) stimulated by a combination of dexamethasone and glucagon. Less synergic stimulation was observed by combining dexamethasone with N6, O2'-dibutyryladenosine 3',5'-monophosphate (dibutyryl-cAMP) or with isoproterenol. The basal level of CPS synthesis in hepatoma cells was higher than that in hepatocytes. CPS synthesis in hepatoma cells was stimulated by dexamethasone and dibutyryl-cAMP but the extent was only 3-fold and 1.8-fold respectively. The synergic effect of combination of dexamethasone and dibutyryl-cAMP was not observed in hepatoma cells. Neither glucagon nor isoproterenol exhibited an appreciable effect on CPS synthesis in hepatoma cells. Insulin and epinephrine suppressed CPS synthesis both in hepatocytes and hepatoma cells. The effect of epinephrine was indicated to be through alpha-adrenergic receptors. The effects of insulin and epinephrine were additive on CPS synthesis both in hepatocytes and hepatoma cells.     

Regulation of phosphoenolpyruvate carboxykinase (GTP) synthesis in rat liver cells. Rapid induction of specific mRNA by glucagon or cyclic AMP and permissive effect of dexamethasone

Isolated rat liver cells maintained in suspension culture for 4 to 5 h synthesize the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase at a rate approximately 5-fold lower than the in vivo hepatic rate. Glucagon rapidly re-induces phosphoenolpyruvate carboxykinase synthesis in such cells. The rate of enzyme synthesis doubles in 40 min and plateaus at a level 6- to 13-fold higher than in control cells 120 min after glucagon addition at maximal concentration. Consistent with the presumed role of cyclic AMP as a mediator of enzyme induction, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, added simultaneously with glucagon, shifts the hormone dose-response curve 2 log units to the left. Moreover, cyclic AMP supplied exogenously to the cells mimics the inductive effect of glucagon. Total cellular RNA isolated from hepatocytes induced by glucagon contains an increased level of mRNA coding for phosphoenolpyruvate carboxykinase, as determined by translational assay. The kinetics and extent of the rise in mRNA level are adequate to explain the stimulation of enzyme synthesis. Although glucagon on its own induces a build-up of phosphoenolpyruvate carboxykinase mRNA and a commensurate stimulation of enzyme synthesis, the glucagon induction is very markedly amplified when the cells are first preincubated with dexamethasone. The glucocorticoid by itself, however, does not have any substantial effect on the level of phosphoenolpyruvate carboxykinase mRNA or on the rate of enzyme synthesis. Its role can therefore be characterized as permissive.     

Regulation of insulin secretion from islets of Langerhans rendered permeable by electric discharge

Hepatocytes were isolated from preweaned neonatal and adult rats and maintained in primary monolayer culture. Cells from preweaned newborns possessed no L-type pyruvate kinase, nor did they synthesize the enzyme. Incubation for 48-72 h in culture medium supplemented with 2 mM-fructose and 0.1 microM-insulin induced the synthesis of L-type pyruvate kinase, as judged by increased enzyme activity and the increased incorporation of [3H]leucine into immunoprecipitable L-type pyruvate kinase. Hepatocytes isolated from 48 h-starved adult rats incorporated less [3H]leucine into L-type pyruvate kinase than did cells isolated from high-carbohydrate-diet-fed rats. The rate of enzyme synthesis by cells from 48 h-starved rats was increased by the inclusion of fructose and insulin in the incubation medium, after a lag phase of 24-48 h. After 4 days in culture in the presence of fructose and insulin, hepatocytes from 48 h-starved rats synthesized L-type pyruvate kinase at similar rates to hepatocytes isolated from high-carbohydrate-diet-fed rats.     

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.     

Regulation and expression of carbamyl phosphate synthetase I mRNA in developing rat liver and Morris hepatoma 5123D

A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitochondrial matrix enzyme carbamyl phosphate synthetase I (Mr = 160,000) was employed to compare relative amounts of the messenger in adult and fetal liver and in Morris hepatoma 5123D and 3924A cells. Northern blot analysis gave a size estimate for the messenger of 6,500-6,700 nucleotides. Carbamyl phosphate synthetase mRNA levels in 15-day-old fetal liver were less than 10% of adult levels; 5123D cells expressed the messenger at levels about 2-fold higher than normal adult liver, but the messenger was undetectable in 3924A cells. Albumin mRNA was also expressed in the former but not in the latter. Maintaining rats for 5 days on a diet containing 60% casein augmented the relative amount of carbamyl phosphate synthetase mRNA by about 2-fold, while a protein-free diet resulted in reduced levels of the mRNA (about 50% compared to animals on a normal diet). Finally, the pattern of hybridization of carbamyl phosphate synthetase cDNA to HindIII-digested genomic DNA showed no differences between normal liver and its corresponding hepatoma; however, a HindIII site polymorphism was observed between Buffalo and ACI rats.     

Expression of carbamoyl-phosphate synthetase I mRNA in Reuber hepatoma H-35 cells. Regulation by glucocorticoid and insulin

Reuber hepatoma H-35 cells actively synthesize the urea cycle enzyme, carbamoyl-phosphate synthetase I. Treatment of H-35 cells with dexamethasone (0.14 microM), however, enhanced synthesis of the enzyme (as measured by incorporation of [35S]methionine) by 4-5-fold. Insulin (0.18 microM) completely inhibited dexamethasone-dependent stimulation of enzyme synthesis. In vitro translation and cDNA hybridization assays were employed to measure effects of dexamethasone plus or minus insulin on levels of mRNA encoding the biosynthetic precursor of carbamoyl-phosphate synthetase I (pCPS) in Reuber H-35 cells. Both measurements yielded similar results: dexamethasone increased pCPS mRNA levels by 4-5-fold and insulin suppressed this response, but only by 50%. Specific cDNA hybridization assays also demonstrated that Reuber H-35 cells, even after hormone treatments, contain only very low levels of albumin mRNA, and no detectable ornithine carbamoyl-transferase mRNA.     

Hormone-sensitive carbohydrate metabolism in rat hepatocyte-hepatoma hybrid cells.

Hepatocyte-hepatoma hybrid cells were obtained by fusion of hepatocytes from adult rats and Fao hepatoma cells in the presence of polyethylene glycol. These hybrids were called hepatocytoma cells. The preservation of liver-specific enzyme activities and metabolic functions was studied in the hybrid clone 1E3. 1) The proliferating hepatocytoma cells formed monolayers presenting morphological similarity to primary cultures of hepatocytes. 2) In contrast to Fao hepatoma cells, activities of all gluconeogenic key enzymes were preserved at normal or reduced levels. 3) Lactate-dependent glucose formation was maintained at a state reduced to 36% of the gluconeogenesis in hepatocytes; no glucose formation was detected in Fao hepatoma cells. 4) The activity of the liver-specific glucokinase was reduced in hepatocytoma cells, but it was still present in contrast to Fao cells. The liver-specific isoenzyme pyruvate kinase type L was replaced by the isoenzyme type M2. 5) Gluconeogenic and glycolytic enzyme activities were regulated in hepatocytoma cells by glucagon (0.1 microM) and by insulin (0.1 microM). 6) The genome of hepatocytoma cells and its expression were stable for at least one year, when spontaneously dedifferentiating cells were removed by recloning in hypoxanthine-aminopterine-thymidine (HAT) medium.     

Effect of prior nutritional status on the activity of lipogenic enzymes in primary monolayer cultures of rat hepatocytes.

Effect of prior nutritional status of the animal on the activity of lipogenic enzymes and the fatty acid content of cultured hepatocytes was investigated. Hepatocytes were isolated from rats that were starved for 24 h ('starved') or continuously fed ('fed'), or starved for 48 h and then re-fed for 48 h ('re-fed') with a carbohydrate-rich fat-free diet, and maintained as monolayer cultures for 96 h in a serum-free glucose-rich medium (Waymouth's MB752/1) supplemented with insulin, dexamethasone and tri-iodothyronine. The fatty acid content and the activities of acetyl-CoA carboxylase, fatty acid synthase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were determined initially at 3 h after plating and then every 24 h. Initially the activities of all the four enzymes were highest in hepatocytes isolated from the re-fed rats and lowest in those from the starved rats. With time in culture, the activity of all these enzymes increased severalfold (2-5, depending on the enzyme under consideration) in hepatocytes isolated from fed and starved rats, whereas there was a severalfold (2-5) decrease in the activity of these enzymes in hepatocytes isolated from re-fed rats. The initial fatty acid content of the hepatocytes from re-fed rats was 2-3 times that in the other two groups of hepatocytes. The fatty acid content seemed to increase in all three groups of hepatocytes during the 96 h in culture, but these apparent increases were not statistically significant.     

Enzymatic,biophysical and ultrastructural changes of plasma membranes in chemical-induced rat hepatoma

Plasma membranes from liver of control rats or from chemical-induced hepatoma were prepared. The basal activity of adenylate cyclase was increased significantly in the rat plasma membranes of DEN-induced hepatoma compared to normal tissue. The glucagon-induced response on the cellular effector systems via guanine nucleotide-binding regulatory proteins (G proteins) was inhibited in hepatoma plasma membranes. These findings suggest that in hepatoma membranes, unlike normal hepatic membranes, the response to hormonal stimuli through regulatory G proteins results in a loss of response to glucagon, as well as to GTP plus glucagon or to GTPγS. However, the activating effects of forskolin, which catalyses the formation of cyclic AMP from ATP acting on the catalytic subunit, were to some extent retained. The methyltransferase-I behaved in the opposite direction to the adenylate cyclase, showing a decreased activity in hepatoma plasma membranes compared to control membranes. In contrast, the activity of the ecto-5′-nucleotidase was significantly increased in hepatoma. These enzymatic changes have been found to influence the membrane fluidity and to be responsible for the ultrastructural modifications of hepatoma plasma membranes which are induced by chemical carcinogens.     

Light form of Morris hepatoma gamma-glutamyltransferase

Using solubilization with bromelain, the light form of gamma-glutamyltransferase was purified from Morris hepatoma 5123D. Some properties of this enzyme were compared to those of the light form rat kidney GGT. Anthglutin and its isomer inhibit competitively the former enzyme but non-competitively the latter. On zymograms of rat control sera, five GGT fractions were noted, but in sera of rats with hepatoma 5123D also the light form and the increase of GGT activity ain region of fraction II were observed. Only these two enzyme fractions react with antibody anti heavy form of Morris hepatoma GGT.     

Developmental and hormonal regulation of carbamoyl-phosphate synthase gene expression in rat liver: evidence for control mechanisms at different levels in the perinatal period

Carbamoyl-phosphate synthase gene expression is found to be primarily regulated by conditions that enhance hepatic glucocorticosteroid levels (hormone injections) and cyclic AMP levels (induction of diabetes). After birth, changes in the level of carbamoyl-phosphate synthase protein follow changes in the level of carbamoylphosphate synthase mRNA, suggesting a pretranslational control mechanism. In fetal rats, carbamoyl-phosphate synthase gene expression is regulated by the same factors as in adults. However, both the level to which carbamoyl-phosphate synthase mRNA can accumulate and the extent to which mRNA can be translated appear to be limited, indicating control mechanisms at the pretranslational and translational level. Finally, in the immediate postnatal period, a transient but pronounced decrease in the rate of degradation of carbamoyl-phosphate synthase protein may play a role in the accumulation of the enzyme.     

Increased expression of N-acetylglucosaminyltransferase-V in human hepatoma cells by retinoic acid and 1alpha,25-dihydroxyvitamin D3

UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6N-acetylglucosaminyltransferase-V activities were determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. When GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-4)(Manbeta1-4GlcNAc-2-amino pyridine (GlcN,GlcN-biant-PA) and UDP-GlcNAc were used as substrates, the enzymes displayed optimum temperatures of 50 degrees C, optimum pHs of 6.5 in each case, K(m) values for UDP-GlcNAc to be 5.8 (Hep3B) and 4.5 mM (HepG2) and K(m) values for GlcN,GlcN-biant-PA (mM) to be 1.28 (Hep3B) and 2.4 (HepG2). This indicates that values of Hep3B GlcNAc-transferase-V were distinguishable with HepG2 enzyme. Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1.423 pmol/(h mg) than that (1.066 pmol/(h mg)) of HepG2. Normal hepatocytes are characterized by very low level of GlcNAc-transferase-V activity whereas hepatoma cells contained high activities. Treatment of hepatoma cells with retinoic acid and 1alpha,2,5-dihydroxyvitamin D(3) (Vit-D(3)) resulted in an increase in GlcNAc-transferase-V activity, while treatment with dimethyl sulfoxide and cytosine-arabinoside resulted in decrease in the enzyme activity. Although retinoic acid (RA) treated cells shows a changed GlcNAc-transferase-V mRNA expression, expression of marker proteins such as alpha-fetoprotein and albumin was not changed. This is the first demonstration of GlcNAc-transferase-V activity in RA and Vit-D(3)-treated hepatoma cell lines.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.     

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.     

Hormonal regulation of two urea-cycle enzymes in cultured foetal hepatocytes.

Foetal-rat hepatocytes were cultured in primary monolayer culture, and activity changes of argininosuccinate synthetase (ASS, EC 6.3.4.5) and argininosuccinase (ASL, EC 4.3.2.1) were followed under defined hormone conditions. In hormone-free medium, cultured cells maintained the enzyme activities at values equal to those of freshly isolated cells for at least 3 days. Continuous addition of dexamethasone produced the development of the two enzyme activities, but only after the first 20h of culture. Under these conditions, urea production by the foetal hepatocytes was concomitantly increased in the culture medium. Pretreatment with dexamethasone for 20h was sufficient to produce the development of ASL activity within the 2 following days. Introduced alone, glucagon induced an increase of ASL activity, but did not affect the ASS activity. The most powerful stimulation of ASS and ASL could be observed in cultured hepatocytes if glucagon and dexamethasone were added simultaneously or sequentially. These results indicated that the development of the receptor complex for the induction of urea-cycle enzymes appears early before birth and established that glucocorticoids amplify the glucagon stimulation of these enzyme activities during foetal life.     

The reduced extracellular calcium requirement for proliferation by neoplastic hepatocytes.

Cells from neonatal rat livers were unable to maintain DNA-synthetic activity in calcium-deficient medium, but neoplastic hepatocytes from Morris hepatomas 5123 tc and 7795 synthesized DNA and proliferated indefinitely in this calcium-deficient medium. The calcium content of fresh hepatoma tissue from which these cultures were derived was as much as 10 times greater than that of normal liver; but this difference could not account for the insensitivity of neoplastic cells to extracellular calcium because it disappeared during subsequent cultivation in vitro.