Alain Fairand

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

The mechanism of action of hydration state was studied on β‐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 β‐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 β‐actin mRNA level in a dose‐dependent manner and induced a stimulation of the β‐actin gene transcription. Thus, cell hydration state regulates gene expression in the liver through a transcriptional mechanism.

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.

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.

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.

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 arginonosuccinate. 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.

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 45 000 M r protein, which decreased in the presence of inhibitors of transcription. A close relationship between the synthesis of the 45 000 M r 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.

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.

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.

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.

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.