Sylvie Renouf

GLUTAMINE AND REGULATION OF GENE EXPRESSION IN RAT HEPATOCYTES : THE ROLE OF CELL SWELLING

Glutamine is able to regulate the expression of various genes in rat hepatocytes. This includes genes coding for proteins involved in glutamine utilization, such as argininosuccinate synthetase (ureagenesis) or phosphoenolpyruvate carboxykinase (gluconeogenesis). Moreover, glutamine is also able to stimulate the expression of genes involved in the acute-phase response, such as the alpha 2-macroglobulin gene. The effect of glutamine on the regulation of gene expression may be explained, at least in part, by the cell swelling due to its sodium-dependent transport. The physiological significance of the effect of glutamine is discussed.

Glutamine and regulation of gene expression in mammmalian cells. Special reference to phosphoenolpyruvate carboxykinase (PEPCK)

The repertoire of the actions of specific amino acids on gene expression is relatively limited in mammalian cells. Glutamine constitutes the most studied amino acid and recent works intended to demonstrate its mechanism of action on two genes: the beta-actin and the phosphoenolpyruvate carboxykinase genes. From these studies, it appears that glutamine may regulate gene expression by, at least, two different mechanisms: one through the glutamine-induced cell swelling, and another through its intracellular metabolism. The involvement of phosphatidylinositol 3-kinase in the signaling pathway triggered by cell swelling is discussed.

Cell swelling increased the α2‐macroglobulin gene expression in cultured rat hepatocytes

The effect of cell swelling on the expression of the α2‐macroglobulin (α2M) gene was studied in hepatocytes in culture. Hypoosmolarity induced an increase (3‐fold increase) in the level of α2M mRNA through a corresponding stimulation of the rate of transcription of the α2M gene. The addition of raffinose (100 mM) corrected the effect of hypoosmolarity at both mRNA and transcriptional level, demonstrating that cell swelling per se was responsible for the observed effect on the expression of the α2M gene. Moreover, the effect of cell swelling was additive to that of interleukin 6, a major mediator of the acute‐phase response.

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.