Regulation of Argininosuccinate Synthetase mRNA Level in Rat Foetal Hepatocytes

Abstract

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.Keywords: argininosuccinate synthetase; mRNA ; gene transcription ; cultured foetal hepatocyte; glucocorticoid.Argininosuccinate synthetase (ASS) catalyses the synthesis of argininosuccinate from aspartate and citrulline, and is one of the key enzymes of the urea cycle in Mammals [l]. This enzyme is expressed in many tissues, with highest expression in the liver and in the kidney, where it plays a role in the arginine synthesis [2], and in the testis [3].Regulation of expression of ASS in the adult rat liver appears complex since increased protein content in the diet, prolonged starvation [4] and administration of glucocorticoids or glucagon have been shown to increase its activity both in vivo [5, 61 and in vitro [7, 81. Most of these treatments also produced an accumulation of specific mRNA [9-111. The expression of this gene was shown to be also regulated by amino acids since it was stimulated by glutamine addition in cultured rat hepatocytes [12] and repressed by arginine in cultured human cellsIn the late foetal period, the urea cycle enzymes are actively synthesized in the liver and this differentiation largely depends on the foetal endocrine state [14-161. Previous studies have shown that the ASS activity appeared in the foetal rat liver during the last days of gestation and that its development depended on the presence of foetal adrenals [14, 151 and on plasma levels of glucagon and insulin [17]. In particular ASS activity did not ~3 1 . The aim of the present work was to characterize the expression of the gene of ASS ...

Expression of the Genes of Arginine-Synthesizing Enzymes in the Rat Kidney during Development

“…Previous works from our laboratory have been directed toward determining the expression of the ASS and ASL genes and identifying the regulatory signals involved in their response in the fetal liver [5][6][7]. This work first presents evidence that the genes coding for ASS and ASL are also transcribed in various extra hepatic tissues during the fetal period.…”

“…Previous studies have focused on the expression of both genes in the developing liver where the corresponding enzyme activities and mRNAs appeared in the late fetal period [3][4][5]. Hormones such as glucocorticoids, glucagon and insulin [5][6][7] were shown to play an essential role in the developmental expression of hepatic ASS and ASL genes. Particularly, glucocorticoids suppression at birth could inhibit the development of the ASL protein and mRNA levels in the newborn liver [6].…”

“…Both enzyme activities have a wide distribution in the adult rat [1] but are predominantly expressed in the liver, kidney and testis, where they are known to synthesize arginine [2]. Previous studies have focused on the expression of both genes in the developing liver where the corresponding enzyme activities and mRNAs appeared in the late fetal period [3][4][5]. Hormones such as glucocorticoids, glucagon and insulin [5][6][7] were shown to play an essential role in the developmental expression of hepatic ASS and ASL genes.…”

Differential Expression Profiling of the Hepatic Proteome in a Rat Model of Dioxin Resistance

“…Such alterations might be viewed as a secondary effect of TCDD because primary TCDD-dependent effects such as the modulation of the concentration of glucocorticoids, glucagons, and insulin are all known to play roles in the liverspecific transcriptional regulation of these proteins (61,62).…”

Glutamine Stimulates Argininosuccinate Synthetase Gene Expression through Cytosolic O-Glycosylation of Sp1 in Caco-2 Cells

“…Total RNA (25 g) was separated onto a 1.5% agarose/formaldehyde gel, transferred onto nylon membranes, and probed with 32 P-labeled ASS cDNA. Hybridizations were performed overnight at 42°C as described previously (21). Blots were then washed and exposed to Hyperfilm using intensifying screens at Ϫ80°C for periods varying from 6 to 72 h. The blots were reprobed with a 18 S ribosomal RNA probe for data normalization.…”