Robert Charles

Inducibility of carbamoylphosphate synthetase (ammonia) in cultures of embryonic hepatocytes: Ontogenesis of the responsiveness to hormones

Glucocorticosteroids and cyclic AMP induce carbamoylphosphate synthetase (ammonia) (CPS) in rat hepatocytes. Using an enzyme immunoassay applied to hepatocyte cultures fixed in situ, it has been demonstrated that the capacity of hepatocytes to synthesize CPS in the presence of both hormones is present as soon as the cells become recognizable as hepatocytes. Immunochemical staining of the cultures shows that hepatocytes do not acquire or express the capacity to accumulate CPS at high rates synchronously. The average levels of CPS per hepatocyte that are observed upon hormone treatment are approx 50-fold lower in embryonic than in adult hepatocytes, corresponding with an approx 10-fold lower synthetic capacity (per gram hepatocytes) and an approx 5-fold smaller size of embryonic compared to adult hepatocytes. Carbamoylphosphate synthetase levels are therefore a good parameter in studies that aim to establish the mechanisms that underly the ontogenesis of the hepatic phenotype.

The initiation of hepatocyte-specific gene expression within embryonic hepatocytes is a stochastic event

To gain insight into the mechanisms that govern the first steps of liver-specific enzyme accumulation upon hormone exposure, the initial accumulation of carbamoylphosphate synthetase, phosphoenolpyruvate carboxykinase, and arginase in monolayer cultures of Embryonic Day 14 rat hepatocytes was studied. By using different fluorescent labels the initial accumulation of two enzymes could be studied simultaneously in individual cells. Both microscopic and flow cytometric analyses showed that the initial expression of genes that are under the same hormonal control appears to lack the coordinated regulation of expression that is seen later in development. The coordination is gradually established during exposure to hormones. Once gene expression becomes coordinated, the enzyme content appears to increase continuously with time. Therefore, we postulate that within individual embryonic hepatocytes the initial intercellular heterogeneity in rate of accumulation of a particular protein may be the result of competition of different genes for an initially limiting supply of common regulatory factors, leading to random differences in the rate of accumulation of the respective gene products. This makes the initiation of liver-specific gene expression within the hepatocytes a stochastic event.

Messenger RNA sorting in enterocytes Co-localization with encoded proteins

This study describes the intracellular compartmentalization of three different mRNAs in the polarized rat fetal enterocyte. They encode proteins that are known to be localized within different regions of the epithelial cell namely (i) the apical, membrane‐bound glycoprotein, lactase‐phlorizin hydrolase (lactase), (ii) the mitochondrially localized enzyme, carbamoylphosphate synthetase (CPS), and (iii) the cytoplasmically localized enzyme, phosphoenolpyruvate carboxykinase (PEPCK). These mRNAs are found in close proximity to their respective protein products, i.e. the apical membrane, mitochondria and cytoplasm, respectively. The significance or these observations is twofold: (i) they indicate that mRNAs are sorted into specific domains of the cytosol of intestinal epithelial cells: and (ii) they imply the presence or two distinct pathways of mRNA targeting one that allows transport of mRNAs that are translated on ribosomes associated with the rough endoplasmic reticulum (lactase mRNA), and the other that allows sorting of mRNAs that are translated on free polysames (CPS and PEPCK mRNA).

Diet- and hormone-induced reversal of the carbamoylphosphate synthetase mRNA gradient in the rat liver lobulus

A hybridocytochemical analysis of adult liver from normal control and from hormonally and dietary‐treated rats was carried out, using radioactively‐labelled probes for the mRNAs of glutamine synthetase (GS), carbamoylphosphate synthetase (CPS) and phosphoenolpyruvate carboxykinase (PEPCK). In line with previous findings, GS mRNA is exclusively expressed in a small pericentral compartment, CPS mRNA exclusively in a contiguous large periportal compartment and PEPCK mRNA across the entire porto‐central distance. The density of labelling in CPS and PEPCK mRNA‐positive hepatocytes decreases in a porto‐central direction. Starvation resulted in a reversal of the gradient of CPS mRNA within its periportal compartment; glucose refeeding counteracted this effect. Livers of glucocorticosteroid‐treated, starved or diabetic rats also revealed a reversal of the normal gradient of CPS mRNA, but now across the entire porto‐central distance. The patterns of expression of GS and PEPCK mRNA remained essentially unchanged, notwithstanding substantial changes in the levels of expression. It is concluded that blood‐borne factors constitute the major determinants for the expression patterns of CPS mRNA within the context of the architecture of the liver lobulus.

The dynamics of the expression of C/EBP mRNA in the adult rat liver lobulus qualifies it as a pericentral mRNA

A hybridocytochemical approach has been applied to establish whether the gene for the C/EBP mRNA might be involved in the topographical regulation of gene expression in adult rat liver. To that end the spatial distribution of the mRNA of C/EBP has been compared to that of the mRNAs of glutamine synthetase (GS), phosphoenolpyruvate carboxykinase (PEPCK) and glucokinase (GK) in normal adult livers, in livers from dcxamethasone‐treated animals and in livers from starved animals refed with glucose for 4 h. In normal rat liver, in situ hybridization with a probe for C/EBP mRNA revealed a low density of apparently homogeneously distributed grains, indicating low levels of C/EBP mRNA. In contrast, the livers of the experimentally‐treated animals revealed a zonal distribution of the mRNA of C/EBP with the highest density of grains around the central venules. The dynamics of the pattern of expression of C/EBP mRNA are virtually identical to that of the GK mRNA. These data qualify C/EBP mRNA as a pericentral mRNA and suggest a role for the C/EBP protein in the topographical regulation of the expression of the GK mRNA.

Pericentral expression pattern of glucokinase mRNA in the rat liver lobulus

The spatial distribution of glucokinase mRNA (GK mRNA) in rat liver was studied by in situ hybridization under normal and inducing conditions. GK mRNA was first detectable in the liver parenchyma of neonatal rats of 1.5 days. The density of grains decreases in a central portal direction. This pattern remains essentially unchanged up to 15 days, after which the adult type of distribution gradually starts to develop, i.e. low density of grains indicating low levels of GK mRNA, in which no gradient of expression could be visualized. Within 2 h after an oral glucose load to starved animals, the GK mRNA expression pattern changed from hardly detectable to a clear gradient with the highest grain density around the terminal central venules. Within 6 h relatively high levels of grains, almost homogeneously distributed across the liver lobule, were observed. Glucocorticosteroid treatment also induced GK mRNA in the pericentral area. It is concluded that the observed induction pattern qualifies GK mRNA as a pericentral mRNA suggesting that the pericentral expression pattern of the protein is primarily regulated at the pretranslational level.

Amino acid environment determines expression of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in embryonic rat hepatocytes

SummaryA completely defined medium (EHM-1), which reflects the amino acid composition of fetal rat serum and contains albumin as the sole proteinaceous compound, allows the accumulation of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in the presence of dexamethasone, dibutyryl cyclic AMP, and triiodothyronine to approximately twice the level attained in a standard culture medium (RPMI 1640) supplemented with 10% fetal bovine serum (and hormones). Using the EHM-1 medium we could show that the capacity of hepatocytes to synthesize phosphoenolpyruvate carboxykinase in the presence of hormones is manifest as soon as the cells differentiate from the embryonic foregut (embryonic Day 11). Furthermore we could show that embryonic hepatocytes can become binuclear or polyploid when cultured in the presence of thyroid hormone.