Expression of the genes of arginine-synthesizing enzymes in the rat kidney during development.

The expression of two genes, coding for argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL), enzymes which synthesize arginine, was studied by Northern analysis in various tissues of fetal rats. The highest expression of both genes was seen in the small intestine, liver and kidney of the fetus. The developmental expression was observed in the kidney where both mRNA levels remained low during the fetal period and they increased concomitantly in both kidney and liver throughout the perinatal life, suggesting that the aptitude to synthesize arginine appeared and developed within the same period in both organs. This developmental activation of the ASS and ASL genes expression corresponded, at least in part, to a transcriptional mechanism in both tissues, as measured by run-on assay. Bilateral adrenalectomy showed that glucocorticoids did not appear to control the developmental expression of both genes in the kidney, in contrast to the situation observed in the liver.

Developmental control of argininosuccinate lyase gene by methylation.

The gene of argininosuccinate lyase (ASL) is expressed in a developmental specific manner in the liver and is regulated by hormones, namely glucocorticoids, glucagon and insulin. To assess the role of DNA methylation in the developmental pattern of ASL gene expression, we analyzed the restriction profile obtained by cleavage of genomic DNA with MspI and HpaII in fetal and adult rat liver, two developmental stages with different levels of expression of the ASL gene. Southern analysis showed that the 5' region of this gene appeared more methylated in the fetal liver which expressed ASL at a low level than in the adult liver where the ASL gene is highly expressed. Moreover, treatment of fetuses of various gestational stages with the hypomethylating agent 5-azacytidine for 18 h caused an increase of the hepatic ASL activity and mRNA level. The stimulating effect of this drug could be also observed in vitro in cultured fetal hepatocytes. These results suggest a developmental control of the ASL gene by the DNA methylation status.

Protein synthesis is involved in the modulation of the level of the phosphoenolpyruvate carboxykinase mRNA by changes in cell volume in isolated rat hepatocytes.

The mechanism of action of hydration state was studied on phosphoenolpyruvate carboxykinase (PCK) gene expression in isolated rat hepatocytes. Hypoosmolarity decreased the level of the PCK mRNA after a lag period of about 60 min. The decreasing effect of hypoosmolarity was totally blocked by inhibitors of both protein synthesis and gene transcription. Moreover, hypoosmolarity specifically increased the synthesis of a 45000 Mr protein, which decreased in the presence of inhibitors of transcription. A close relationship between the synthesis of the 45000 Mr protein and the decrease in the PCK mRNA level was observed, suggesting that this protein might potentially be involved in the regulation of the level of the PCK mRNA by cell swelling.

Regulation of argininosuccinate synthetase mRNA level in rat foetal hepatocytes.

Expression of the hepatic gene for argininosuccinate synthase (ASS), one of the key enzymes of the urea cycle, was analysed during the perinatal period in the rat. To this end, the amount of specific mRNA was measured in the liver at various stages of development and in cultured foetal hepatocytes maintained in different hormonal conditions. The ASS mRNA was first detected in 15.5-day foetuses and its level increased concomitantly with a rise in the enzyme activity, suggesting that the appearance of the ASS activity reflects the turning on of specific gene transcription. This was demonstrated by run-on assay which showed an enhanced rate of transcription of the ASS gene during the perinatal period. When foetal hepatocytes were cultured with dexamethasone, a dose-dependent increase in ASS mRNA was measured, which was completely abolished by actinomycin D addition. The transcription rate of the gene was increased about twofold in the presence of the steroid, as measured by nuclear run-on assay. This transcriptional action could additionally require a protein factor since it could be inhibited by the simultaneous addition of puromycin. Insulin or glucagon respectively repressed or enhanced the dexamethasone-induced accumulation of ASS mRNA when added simultaneously with the steroid for 24 h. This developmental regulation of the ASS mRNA by glucocorticoids, insulin and glucagon could account for the modulation of the enzyme activity previously observed in vivo and in vitro in the foetal liver.

Glutamine inhibits the lowering effect of glucose on the level of phosphoenolpyruvate carboxykinase mRNA in isolated rat hepatocytes.

The expression of phosphoenolpyruvate carboxykinase (P-pyruvate CK) was shown to be decreased by hypoosmolarity and increased by glutamine in perfused liver from fed rats [Newsome, W. P., Warskulat, U., Noe, B., Wettstein, M., Stoll, B., Gerok, W. & Häussinger, D. (1994) Biochem, J. 304, 555-560]. This work was undertaken to specify the mechanisms of glutamine action, using isolated hepatocytes from rats that had been starved for 24 h. At low concentrations (up to 5 mM), glutamine elicited a decrease in the level of P-pyruvate CK mRNA through cell swelling and, at higher concentrations, an increase in the mRNA level was observed. Experiments with combinations of glucose and glutamine or glucose and various amino acids demonstrated that glutamine counteracted the inhibitory effect of glucose on P-pyruvate CK mRNA at a transcriptional, level, and strongly suggested that the amide group of glutamine was involved in this effect. The metabolism of glucose was required for the reinforcement of the apparent stimulatory effect of glutamine, as demonstrated by the use of various sugars. Glucosamine, but not mannosamine, increased the level of P-pyruvate CK mRNA, as did glucose plus glutamine. These results suggest that the pathway leading from glucosamine-6-phosphate production might be responsible, at least partly, for the effect observed on P-pyruvate CK mRNA.

Hypoosmolarity and glutamine increased the beta-actin gene transcription in isolated rat hepatocytes.

The mechanism of action of hydration state was studied on beta-actin gene expression in isolated hepatocytes. Results obtained with Northern blot analysis and run on transcription assays show that hypoosmolarity increased and hyperosmolarity decreased the beta-actin mRNA level through a corresponding modulation of the rate of the gene transcription. Glutamine, which is known to induce cell swelling, also increased the beta-actin mRNA level in a dose-dependent manner and induced a stimulation of the beta-actin gene transcription. Thus, cell hydration state regulates gene expression in the liver through a transcriptional mechanism.

Regulation of argininosuccinate synthetase level by corticosteroid and pancreatic hormones during perinatal period.

The urea cycle takes place in the hepatocyte of ureothelic animals. The conversion of ammonia into urea involves five reactions. The first 2 take place in the matrix of the mitochondria, the last 2 occur in the cytosol. Argininosuccinate synthetase (AS) is the third reaction of the urea cycle. It catalyses the condensation of citrulline and aspartate into argininosuccinate. We have previously reported that rat AS activity was present in the cytosol and the outer membrane of the mitochondria. We have shown that, at the activity level, the colocation of AS was changing during fetal and neonatal development and was under the control of corticosteroid and pancreatic hormones. However, an unresolved issue was whether both AS had the same specific activity and that their location was changing during ontogenesis or that the specific activities of mitochondrial and cytosolic enzymes were different and/or modified during this period. In the present report, we compared the compartmentalization of AS activity and protein level in the fetus, the new-born and the adult rat and the role of corticosteroid and pancreatic hormones. Specific activities of both AS remained unchanged during ontogenesis. Glucocorticoids induced an increase in mitochondrial AS while glucagon appeared to induce a concomitant decrease in the level of mitochondrial AS and an increase in cytosolic AS.

Glucocorticoid-dependent induction of the mRNA coding for argininosuccinate lyase in cultured fetal rat hepatocytes.

Dexamethasone increased both argininosuccinate lyase (ASL) activity and specific mRNA level in cultured fetal hepatocytes. Addition of various inhibitors of RNA synthesis showed that the increase in ASL mRNA may be related to an enhancement of ASL gene transcription, but not to a specific messenger stabilization. An apparent half-life of about 12 h for ASL mRNA was found in both untreated and dexamethasone-treated hepatocytes. About 30 h were necessary to observe the maximal effect of dexamethasone, and, in addition, both puromycin and cycloheximide (two inhibitors of protein synthesis) blocked the inducing effect of the steroid. These results suggested the involvement of intermediary protein(s) in the mechanism of induction of ASL mRNA by glucocorticoids.

Demonstration of argininosuccinate synthetase activity associated with mitochondrial membrane: characterization and hormonal regulation.

Argininosuccinate synthetase (AS) is the third enzyme in ureogenesis, it catalyses the reaction of condensation of citrulline and aspartate into argininosuccinate. In the present report, we described the first characterization of AS within the outer membrane of rat liver mitochondria. Mitochondria-associated AS displayed the same kinetic characteristics as the cytoplasmic enzyme, but was found to be thermostable while cytoplasmic AS was not. The evolution of the co-location of AS was analyzed during ontogenesis. Total AS activity increased throughout rat fetal development. Simultaneously, the subcellular distribution of the enzyme has changed. AS activity was mainly mitochondrial in fetal and new-born liver liver and cytoplasmic in adult rat liver. The variation in subcellular distribution of AS may be due to the dramatic changes in hormonal levels that occur during this period. The role of corticosteroid and pancreatic hormones was studied. During fetal period, corticosteroid hormones induced an increase in mitochondria-associated AS activity. This was prevented by insulin. Glucagon did not modify total AS activity but reduced mitochondrial AS activity, meanwhile, a comparable increase in cytoplasmic AS activity was observed. One may hypothesize that glucagon may participate in the transfer of mitochondrial enzyme into the cytosol.

Changes in levels of argininosuccinate lyase mRNA during induction by glucagon and cyclic AMP in cultured foetal-rat hepatocytes.

During the perinatal period, the activity of the urea-cycle enzyme argininosuccinate lyase (ASL) is regulated by glucocorticoids, glucagon and insulin. In this study, the effects of glucagon and cyclic AMP (cAMP) analogues were examined on the synthesis of ASL and on the level of its corresponding mRNA in cultured foetal hepatocytes. Northern-blot analysis revealed that these agents only gave a transient induction of ASL mRNA amount, which reached a peak at 6 h and declined thereafter. This induction preceded the increase in enzyme activity and amount which could be observed for 2 or 3 days of culture. Stimulation of ASL mRNA accumulation by a combination of cAMP analogues and dexamethasone was additive, indicating that glucocorticoids and cAMP are both necessary to promote hepatocyte differentiation and that inductions could occur via independent pathways. Induction by cAMP analogues could be abolished by actinomycin D, suggesting a control mechanism at the transcriptional level. Puromycin was without effect on ASL mRNA induction by cAMP, indicating that no ongoing protein synthesis was required in the stimulation process.

Changes in argininosuccinate lyase gene expression in the rat liver during development.

Expression of the hepatic enzyme argininosuccinate lyase (ASL), one of the urea cycle enzymes, was analyzed during the perinatal period in the rat. To this end, ASL was purified, an ELISA assay was established to quantify the enzyme protein and a cDNA clone was used to measure the amount of specific mRNA in the liver in various stages of development. During the last few days of fetal life, both enzyme and hybridizable RNA were present at levels far below those measured in the fully differentiated adult liver. Just after birth, they increased rapidly and the mRNA accumulation, particularly, could result from an enhanced rate of transcription as suggested by the experiment with actinomycin D. This postnatal shift in ASL expression was also linked to adrenal activation at birth, as shown by adrenalectomy. However, the extent to which the ASL protein accumulated after birth appeared to be limited when compared to mRNA accumulation, suggesting control mechanisms at the translational level. Thus, during the perinatal period of the rat, both transcriptional and translational control might be implicated in the expression of the ASL gene.

Development and hormonal control of thioredoxin and the thioredoxin-reductase system in the rat liver during the perinatal period.

The development and hormonal regulation of thioredoxin and of the thioredoxin-reductase system were investigated during the perinatal period in rat liver. An immunological procedure was developed in order to quantify thioredoxin in fetal and neonatal hepatocytes. Both immunoreactive thioredoxin and thioredoxin-reductase activity appeared on day 16.5 of pregnancy. The level of immunoreactive thioredoxin increased during the late fetal period, and its level was the same 24 h after birth. Moreover, its development was not subjected to hormonal regulation by corticosteroids and glucagon. In contrast, thioredoxin-reductase activity increased 3 times during the late fetal period and presented a marked increase 24 h after birth. In the absence of glucocorticoids there was no increase in the level of thioredoxin reductase, while administration of hydrocortisone acetate and glucagon to fetuses prematurely evoked its activity. This study suggests that if thioredoxin acts physiologically, this activity is related to the state of reduction of the molecule rather than to the total concentration in the liver.

Expression of argininosuccinate lyase mRNA in foetal hepatocytes. Regulation by glucocorticoids and insulin.

Argininosuccinate lyase (ASL), the fourth enzyme of the urea cycle, belongs to a group of liver enzymes appearing in the late foetal period in the rat. Several hormones, including glucocorticosteroids and insulin have been implicated in the control of the development of this enzyme activity. In this study, the cloned cDNA was used to measure the relative abundance of ASL mRNA in the livers of rats at various stages of perinatal development and in cultured foetal hepatocytes during hormonal manipulations. The ASL mRNA was first detectable on day 15.5 of gestation and increased in amount concomitantly with the rise in the enzyme activity, suggesting that the appearance of enzyme activity reflects the turning on of specific gene transcription. When foetal hepatocytes were exposed to dexamethasone, an increase in ASL mRNA was detected, which was completely abolished by addition of actinomycin D, suggesting a transcriptional effect of the steroid. In contrast, administration of cortisol to foetuses in utero had no effect on the mRNA level, suggesting that the steroid action is inhibited in the intra-uterine environment. Insulin might be the inhibiting factor since it completely repressed the dexamethasone-induced accumulation of ASL mRNA in foetal hepatocytes. These data were confirmed in vivo by experiments using streptozotocin, which produces insulin-depleted foetuses and causes the accumulation of ASL mRNA. This regulation of ASL mRNA by glucocorticoids and insulin could account for the modulation of the enzyme activity observed in vivo and in vitro.

Effects of pancreatic hormones and glucocorticosteroids on argininosuccinate synthetase and argininosuccinase activities of rat liver during the perinatal period: in vivo and in vitro studies.

The activity changes of two urea cycle enzymes, argininosuccinate synthetase (ASS) and argininosuccinase (ASL), were followed after corticosteroid and pancreatic hormone treatments in utero and in primary cultured fetal hepatocytes. The ASL activity which was induced by glucagon or by (Bu)2cAMP administration was enhanced by a treatment with streptozotocin for 2 days, although ASS was not changed under these conditions. The activity of both enzymes was enhanced by cortisol administration in utero only in streptozotocin-treated fetuses, suggesting an inhibitory effect of insulin. In cultured fetal hepatocytes, dexamethasone produced a marked increase of the two enzyme activities, which was abolished by the simultaneous addition of insulin. The parallel results obtained with these two experimental models allow one to conclude that the high plasma insulin level in late gestation might repress the development of ASS and ASL activities in utero and antagonize the effect of corticosteroids on these enzyme activities.

Regulation of ureagenesis in neonatal rat liver: influence of cycloheximide in vivo and in vitro.

In order to establish whether the postnatal rise in five urea cycle enzyme activities is associated with protein synthesis, the effects of cycloheximide on urea cycle enzyme activities, citrulline and urea productions in vivo and in vitro were studied. A single injection of cycloheximide (10 microgram) 9 h after birth significantly prevented the rise in enzyme activities and citrulline concentration which normally occurred after birth, while the urea level remained unchanged. These data suggest that the postnatal rise in urea cycle enzyme activities might be associated with a protein synthesis. The rate of citrulline synthesis by liver slices was reduced (about 50%) in cycloheximide-treated neonatal liver, while the rate of urea production was not significantly decreased. The results obtained in vivo are in good agreement with in vitro experiments: citrullinogenesis was only affected by cycloheximide treatment. When cycloheximide 10 mM was added to the incubation medium, the ability of control neonatal liver slices to produce citrulline and urea was reduced to 42 and 11%, respectively. Since argininosuccinic acid addition in the medium did not produce a rise in urea synthesis, it is concluded that argininosuccinase is most responsive after incubation in cycloheximide and therefore becomes the rate-limiting step of the urea synthesis.

Control of urea synthesis in fetal rat liver slices.

Urea synthesis in fetal rats was studied using a liver slice system with ammonium chloride as nitrogen source. In term fetuses, the rate of urea formation increases with ammonium chloride concentration up to 100 microM and is enhanced by the addition of ornithine. The developmental pattern for urea synthesis is very similar to that found in the enzyme activity studies: urea production increases slightly during fetal life and then rises rapidly at birth. It is found that fetal liver has an absolute requirement for glucocorticosteroids to develop a normal urea synthesis. The accordance with argininosuccinate synthetase activity measured in liver homogenate is discussed.

[Development and control of liver amylo-1,6-glucosidase activity in the fetal rat]. / Evolution et controle de l'activité de l'amylo-1,6-glucosidase du foie foetal de rat.

The development of amylo-1,6-glucosidase activity is studied in fetal rat liver. The activity of control fetuses is high on day 17.5, decreases from day 17.5 to day 19.5, and then rises during the next days. In hypophysectomised fetuses, the increase of the activity is suppressed but not the decrease. Moreover, if the mother is adrenalectomized the decrease and the increase are abolished in hypophysectomised fetuses. Growth hormone administration is quite effective in preventing the decrease in enzyme activity but cortisol treatment does not prevent it. In contrast, cortisol produces a precocious decrease of the activity in intact fetuses. These findings suggest that during fetal life, two hormonal regulation mechanisms are involved in the regulation of amylo-1,6-glucosidase activity: cortisol has a repressive effect on the enzymic activity while growth hormone acts as an inducer.