Complementary expression of glutamine synthetase and carbamoylphosphate synthetase I in ornithine carbamoyltransferase-deficient mouse liver (spf–ash mouse)

 Glutamine synthetase and carbamoylphosphate synthetase I expression was examined immunohistochemically in livers of spf–ash homozygous and hemizygous mice, in which one of the urea cycle enzymes (ornithine carbamoyltransferase) is deficient and hyperammonemic disorders are obvious. In the mutant adult mouse liver, only hepatocytes lining central veins expressed glutamine synthetase. In contrast, other hepatocytes expressed carbamoylphosphate synthetase I but not glutamine synthetase. This complementary expression pattern is similar to that seen in wild-type mouse liver. In the liver of mutant young mice, which showed severe retarded growth and abnormal hair and skin development, the developmental expression pattern of both enzymes was also similar to that of the corresponding wild-type liver. However, suppression of carbamoylphosphate synthetase I expression in the pericentral hepatocytes occurred later in the mutant than in wild-type liver. These results show that high plasma concentrations of ammonium ions, which are one of the substrates for both the enzymes, do not change their complementary expression. Instead they support the idea that factor(s) associated with central veins rather than humoral factors direct pericentral hepatocytes to express glutamine synthetase and to suppress carbamoylphosphate synthetase I expression. Accepted: 24 April 1997     

Expression patterns of mRNAs for ammoniametabolizing enzymes in the developing rat: the ontogenesis of hepatocyte heterogeneity

Summary The expression patterns of the mRNAs for the ammonia-metabolizing enzymes carbamoylphosphate synthetase (CPS), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were studied in developing pre- and neonatal rat liver byin situ hybridization.In the period of 11 to 14 embryonic days (ED) the concentrations of GS and GDH mRNA increases rapidly in the liver, whereas a substantial rise of CPS mRNA in the liver does not occur until ED 18. Hepatocyte heterogeneity related to the vascular architecture can first be observed at ED 18 for GS mRNA, at ED 20 for GDH mRNA and three days after birth for CPS mRNA. The adult phenotype is gradually established during the second neonatal week, i.e. GS mRNA becomes confined to a pericentral compartment of one to two hepatocytes thickness, CPS mRNA to a large periportal compartment being no longer expressed in the pericentral compartment and GDH mRNA is expressed over the entire porto-central distance, decreasing in concentration going from central to portal. Comparison of the observed mRNA distribution patterns in the perinatal liver, with published data on the distribution of the respective proteins, points to the occurrence of posttranslational, in addition to pretranslational control mechanisms in the period of ontogenesis of hepatocyte heterogeneity.Interestingly, during development all three mRNAS are expressed outside the liver to a considerable extent and in a highly specific way, indicating that several organs are involved in the developmentally regulated expression of the mRNAs for the ammonia-metabolizing enzymes, that were hitherto not recognized as such.     

Development of enzymic zonation in liver parenchyma is related to development of acinar architecture

The appearance of the distribution patterns of the NH3-metabolizing enzymes carbamoylphosphate synthetase, glutamate dehydrogenase, and glutamine synthetase in the developing liver of an altricial species (rat) was compared with that in the developing liver of a closely related, precocial species (spiny mouse). The comparison showed that the development of hepatic acinar architecture, rather than perinatal adaptation, is responsible for the development of periportal and pericentral compartments of gene expression. Conditions that confine the expression of specific enzymes to the pericentral compartment of the acinus originate before conditions that confine the expression of (other) specific enzymes to the periportal compartment. However, whether or not the site of gene expression is restricted to specific compartments within the liver acinus, the rate of expression of the gene involved can also be adaptively regulated. Therefore, different factors appear to control the site and the rate of gene expression within one tissue.     

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.     

Complementary distribution of carbamoylphosphate synthetase (ammonia) and glutamine synthetase in rat liver acinus is regulated at a pretranslational level

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.     

Heterogeneous expression of glutamine synthetase mRNA in rat liver parenchyma revealed by in situ hybridization and Northern blot analysis of RNA from periportal and perivenous hepatocytes

Using radiolabeled specific cDNA glutamine synthetase mRNA could be detected by in situ hybridization exclusively within those few perivenous hepatocytes which stained immunocytochemically for glutamine synthetase. This localization of glutamine synthetase mRNA was recently reported by Moorman et al. [(1988) J. Histochem. Cytochem. 36, 751-755]. Biotinylated cDNA was not suitable for mRNA detection because of a very high background staining under the conditions of in situ hybridization. Dot blot and Northern blot analysis of RNA isolated from periportal and perivenous subfractions of hepatocytes also demonstrated the exclusive perivenous localization of two hybridizable glutamine synthetase mRNAs of length 2.8 and 1.6 kilobases. These results indicate that the unique heterogeneity of glutamine synthetase in rat liver parenchyma is controlled at the pretranslational level.     

Induction of glutamine synthetase and transient co-expression with carbamoylphosphate synthetase in hepatocytes transplanted into fat pads of syngeneic hosts

Isolated rat hepatocytes were transplanted into the interscapular and both anterior lateral fat pads of hepatectomized syngeneic rats. At various time points following transplantation, the fat pads were removed, fixed and embedded in paraffin. Serial sections were stained for glutamine synthetase (GS) and carbamoylphosphate synthetase (CPS) using specific antisera and the PAP technique. The initially low fraction of GS+-heptatocytes remained low up to the fourth day, then increased strikingly up to almost 100% and declined gradually after the 14th day. In contrast, the number of CPS+-cells declined continuously to about 30% after 28 days. If the animals were exposed to CCl4 prior to the isolation of the hepatocytes in order to reduce the number of GS+-cells in the initial cell suspension similar results were obtained and no difference in the probability of the colony formation was noted between this and the normal hepatocyte suspensions indicating that the appearance of the GS+-phenotype was not due to a selective survival of these cells. Analysis of the staining intensity of the transplanted hepatocytes revealed the appearance of two populations of GS+-hepatocytes, one with a strong and one with a weak staining, during the course of formation of larger nodules, while only a single weakly stained population could be discerned with respect to the staining for CPS. These results demonstrate that all hepatocytes or at least their descendents can be induced to express GS by the environmental conditions of the fat pads, and that GS and CPS can be co-expressed with an apparently reciprocal relationship.     

Hepatocyte heterogeneity in the metabolism of amino acids and ammonia.

With respect to hepatocyte heterogeneity in ammonia and amino acid metabolism, two different patterns of sublobular gene expression are distinguished: 'gradient-type' and 'strict- or compartment-type' zonation. An example for strict-type zonation is the reciprocal distribution of carbamoylphosphate synthase and glutamine synthase in the liver lobule. The mechanisms underlying the different sublobular gene expressions are not yet settled but may involve the development of hepatic architecture, innervation, blood-borne hormonal and metabolic factors. The periportal zone is characterized by a high capacity for uptake and catabolism of amino acids (except glutamate and aspartate) as well as for urea synthesis and gluconeogenesis. On the other hand, glutamine synthesis, ornithine transamination and the uptake of vascular glutamate, aspartate, malate and alpha-ketoglutarate are restricted to a small perivenous hepatocyte population. Accordingly, in the intact liver lobule the major pathways for ammonia detoxication, urea and glutamine synthesis, are anatomically switched behind each other and represent in functional terms the sequence of the periportal low affinity system (urea synthesis) and a previous high affinity system (glutamine synthesis) for ammonia detoxication. Perivenous glutamine synthase-containing hepatocytes ('scavenger cells') act as a high affinity scavenger for the ammonia, which escapes the more upstream urea-synthesizing compartment. Periportal glutaminase acts as a pH- and hormone-modulated ammonia-amplifying system in the mitochondria of periportal hepatocytes. The activity of this amplifying system is one crucial determinant for flux through the urea cycle in view of the high Km (ammonia) of carbamoylphosphate synthase, the rate-controlling enzyme of the urea cycle. The structural and functional organization of glutamine and ammonia-metabolizing pathways in the liver lobule provides one basis for the understanding of a hepatic role in systemic acid base homeostasis. Urea synthesis is a major pathway for irreversible removal of metabolically generated bicarbonate. The lobular organization enables the adjustment of the urea cycle flux and accordingly the rate of irreversible hepatic bicarbonate elimination to the needs of the systemic acid base situation, without the threat of hyperammonemia.     

Induction of glutamine synthetase and transient co-expression with carbamoylphosphate synthetase in hepatocytes transplanted into fat pads of syngeneic hosts

Summary Isolated rat hepatocytes were transplanted into the interscapular and both anterior lateral fat pads of hepatectomized syngeneic rats. At various time points following transplantation, the fat pads were removed, fixed and embedded in paraffin. Serial sections were stained for glutamine synthetase (GS) and carbamoylphosphate synthetase (CPS) using specific antisera and the PAP technique. The initially low fraction of GS⁺-heptatocytes remained low up to the fourth day, then increased strikingly up to almost 100% and declined gradually after the 14th day. In contrast, the number of CPS⁺-cells declined continuously to about 30% after 28 days. If the animals were exposed to CCl₄ prior to the isolation of the hepatocytes in order to reduce the number of GS⁺-cells in the initial cell suspension similar results were obtained and no difference in the probability of the colony formation was noted between this and the normal hepatocyte suspensions indicating that the appearance of the GS⁺-phenotype was not due to a selective survival of these cells. Analysis of the staining intensity of the transplanted hepatocytes revealed the appearance of two populations of GS⁺-hepatocytes, one with a strong and one with a weak staining, during the course of formation of larger nodules, while only a single weakly stained population could be discerned with respect to the staining for CPS. These results demonstrate that all hepatocytes or at least their descendents can be induced to express GS by the environmental conditions of the fat pads, and that GS and CPS can be co-expressed with an apparently reciprocal relationship.     

Expression of carbamoylphosphate synthetase I and glutamine synthetase in hepatic organoids reconstructed by rat small hepatocytes and hepatic nonparenchymal cells

The objective of this study was to investigate the expression of carbamoylphosphate synthetase I (CPS) and glutamine synthetase (GS) in small hepatocyte colonies and whether the heterogeneous expression of the enzymes could be induced during the maturation of small hepatocytes. Small hepatocytes isolated from an adult rat liver were cultured and proliferated to form colonies. The expression of CPS and GS was examined using immunocytochemistry and immunoblotting. In this culture more than 99% of morphologically hepatic cells were positive for CPS and all small hepatocytes were negative for GS at day 5. CPS-positive cells dramatically decreased with time in culture, whereas GS-positive ones appeared and their number increased in the colonies. Two to 3 weeks after plating, colonies with rising and piled-up cells appeared and the number of such colonies reached about 25% of all colonies at day 30. In most rising and piled-up cells in colonies both proteins were strongly expressed, whereas many small hepatocytes in monolayer colonies did not express either protein. When small hepatocytes in monolayer colonies were overlayed with Matrigel, the cells gradually piled up and both CPS and GS proteins were dramatically induced. The expression of CPS and GS in small hepatocytes may interact with the extracellular matrix because the rising and piled-up cells appear to be induced by the extracellular matrix produced by hepatic nonparenchymal cells.     

Expression of glutamine synthetase in macrophages.

We studied the expression of glutamine synthetase in liver macrophages (Kupffer cells, KCs) in situ and in culture. Glutamine synthetase was detectable at the mRNA and protein level in freshly isolated and short-term-cultured rat liver macrophages. Enzyme activity and protein content were about 9% of that in liver parenchymal cells. In contrast, glutamine synthetase mRNA levels in liver macrophages apparently exceeded those in parenchymal liver cells (PCs). By use of confocal laser scanning microscopy and specific macrophage markers, immunoreactive glutamine synthetase was localized to macrophages in normal rat liver and normal human liver in situ. All liver macrophages stained positive for glutamine synthetase. In addition, macrophages in rat pancreas contained immunoreactive glutamine synthetase, whereas glutamine synthetase was not detectable at the mRNA and protein level in blood monocytes and RAW 264.7 mouse macrophages. No significant amounts of glutamine synthetase were found in isolated rat liver sinusoidal endothelial cells (SECs). The data suggest a constitutive expression of glutamine synthetase not only, as previously believed, in perivenous liver parenchymal cells but also in resident liver macrophages.     

FATTY ACID SYNTHETASE OF BRAIN: DEVELOPMENT, INFLUENCE OF NUTRITIONAL AND HORMONAL FACTORS AND COMPARISON WITH LIVER ENZYME

Abstract— The activity of fatty acid synthetase was studied in the brain and liver of the developing rat. Synthetase activity in brain was considerably higher in foetal and suckling rats than in older animals However, except for a small transient rise in the perinatal period, activity in liver was low until weaning when a dramatic rise occurred. Activity in brain varied according to the quantity of dietary fat only in long-term experiments, whereas in liver nutritional influences clearly predominated in determining the rapid developmental changes of synthetase activity. Administration of hydrocortisone diminished hepatic activity but did not change brain synthetase. In the hypothyroid state activity in brain and liver was consistently decreased. However, in the hyperthyroid state hepatic activity increased but activity in brain did not change. The relatively high activity of fatty acid synthetase during brain development has been discussed in relation to the critical role of this enzyme system in brain metabolism. The effect of the hypothyroid state on the activity of brain synthetase suggests the possibility of hormonal control of this enzyme activity. The responses of hepatic synthetase to the hormonal influences delineate a specific step by which these compounds may exert their effect on fatty acid biosynthesis.     

Regulation of mRNA levels of rat liver carbamoylphosphate synthetase by glucocorticosteroids and cyclic AMP as estimated with a specific cDNA

The construction and cloning of a cDNA complementary to the mRNA of rat liver carbamoylphosphate synthetase (ammonia) is described. Using this cDNA, the size of the mature, cytosolic carbamoylphosphate synthetase (ammonia) mRNA is estimated to be 6.0 Kb. The levels of carbamoylphosphate synthetase (ammonia) mRNA in liver are shown to be regulated by glucocorticosteroids and cyclic AMP. By studying mRNA levels of carbamoylphosphate synthetase, albumin and phosphoenolpyruvate carboxykinase, using specific cDNA clones, we show that carbamoylphosphate synthetase gene expression, like that of albumin is liver-specific.     

Submitochondrial localization and function of enzymes of glutamine metabolism in avian liver

Glutamine synthetase (EC 6.3.1.2) was localized within the matrix compartment of avian liver mitochondria. The submitochondrial localization of this enzyme was determined by the digitonin-Lubrol method of Schnaitman and Greenawalt (35). The matrix fraction contained over 74% of the glutamine synthetase activity and the major proportion of the matirx marker enzymes, malate dehydrogenase (71%), NADP-dependent isocitrate dehydrogenase (83%), and glutamate dehydrogenase (57%). The highest specific activities of these enzymes were also found in the matrix compartment. Oxidation of glutamine by avian liver mitochondria was substantially less than that of glutamate. Bromofuroate, an inhibitor of glutamate dehydrogenase, blocked oxidation of glutamate and of glutamine whereas aminoxyacetate, a transaminase inhibitor, had little or no effect with either substrate. These results indicate that glutamine metabolism is probably initiated by the conversion of glutamine to glutamate rather than to an alpha-keto acid. The localization of a glutaminase activity within avian liver mitochondria plus the absence of an active mitochondrial glutamine transaminase is consistent with the differential effects of the transaminase and glutamate dehydrogenase inhibitors. The high glutamine synthetase activity (40:1) suggests that mitochondrial catabolism of glutamine is minimal, freeing most of the glutamine synthesized for purine (uric acid) biosynthesis.     

Acquisition of antigens characteristic of adult pericentral hepatocytes by differentiating fetal hepatoblasts in vitro

Antigens specific to pericentral hepatocytes have been studied in adult mouse liver, during fetal development, and in cultured fetal hepatoblasts. Antibody reactive with glutamine synthetase stained all fetal liver cells but almost all cells lost this antigen after birth; only a single layer of pericentral cells retained it in adulthood. In contrast, monoclonal antibodies to major urinary protein (MUP) did not detect the antigen until approximately 3 wk after birth, after which time the cells within 6-10 cell diameters of the central veins were positive. Cultured fetal liver cells from embryos at 13 +/- 1 d of gestation were capable of differentiating in vitro to mimic events that would occur had the cells remained in the animal. About 10-20% of the explanted cells grew into clusters of hepatocyte-like cells, all of which stained with albumin antibodies. MUP monoclonals were reactive with one-half of the differentiated fetal hepatocytes. Glutamine synthetase was present in all hepatocytes after several days in culture and gradually decreased and remained in only occasional cells, all of which also contained the MUP antigen. These findings suggest that a sequence of gene controls characterizes expression of specific genes in developing liver, and that differentiating fetal hepatoblasts are capable of undergoing similar patterns of gene activity in culture.     

The initial accumulation of carbamoylphosphate synthetase in embryonic rat hepatocytes, and the cell cycle

The hormone-induced expression of the hepatocyte-specific enzyme carbamoylphosphate synthetase can take place in each phase of the cell cycle and is not restricted to the G1 or the G0 phase. To arrive at this conclusion, the cell cycle parameters of embryonic day 14 rat hepatocytes in vitro were determined by autoradiography after labeling with (3H)-TdR or with (3H)- and (14C)-TdR. An S-phase of approximately 14 h, a G2 + M-phase of 8 h, a G1-phase of 8-13 h and a total cell cycle of 30-35 h were measured. Freshly isolated embryonic hepatocytes have exponential growth parameter values, but shift to a steady state growth under culture conditions in the presence of hormones (glucocorticosteroids, thyroid hormones and cyclic AMP). The length of the S-phase and of the total cell cycle remain constant during the culture time. The time course of accumulation of carbamoylphosphate synthetase protein in embryonic hepatocytes is identical in all phases of the cell cycle. It is suggested that hormones, in particular glucocorticosteroids, simultaneously and independently regulate growth mode and gene expression in developing hepatocytes. The nucleotide-analogue 5-bromodeoxyuridine inhibits the hormone-induced expression of carbamoylphosphate synthetase only in cells that are exposed to the drug during early S-phase, indicating replication of the carbamoylphosphate synthetase gene in that part of the cell cycle.     

Reorganization of the endocytic compartment in regenerating liver.

Antibodies raised to two membrane proteins present in rat liver endosomal fractions were used to study changes occurring in the endocytic compartment of hepatocytes during liver regeneration. Antibodies to the 42-kDa subunit (RHL-1) of the asialoglycoprotein receptor showed, by Western blotting of liver microsomes and endosomes, that there was a reduced expression of the receptor in liver 24 h following a partial hepatectomy. Immunocytochemical staining of thin sections of regenerating livers using these antibodies indicated that there was an intracellular relocation of endocytic structures in hepatocytes. The two main endocytic regions immunocytochemically stained in normal liver--one located beneath the sinusoidal plasma membrane and the other abutting the bile canaliculus--were replaced, in regenerating liver, by staining more closely associated with a region underlying the baso-lateral plasma membrane. A 140-kDa pI 4.3 calmodulin-binding protein located in endocytic and plasma membranes was also demonstrated, using a radio-iodinated calmodulin-binding assay, to be present at reduced levels in endosomes isolated from regenerating livers. Antibodies to this calmodulin-binding protein stained the hepatocyte's cytoplasm in a punctate manner. However, in regenerating liver, the staining was located in regions underlying the baso-lateral and apical plasma membrane of hepatocytes. Together, the results demonstrate that a reorganization of the endocytic compartment has occurred in hepatocytes 24 h following hepatectomy, with two endosomal proteins becoming relocated to a region below the baso-lateral-apical surface regions of hepatocytes.     

Low asialoglycoprotein receptor expression as markers for highly proliferative potential hepatocytes.

Development of a reliable method to isolate highly proliferative potential hepatocytes will provide insight into the molecular mechanisms of liver regeneration, as well as proving crucial for the development of a biohybrid artificial liver. The aim of this study is to isolate highly proliferative, e.g., progenitor-like, hepatocytes. To this end, we fractionated hepatocytes expressing low and high levels of the asialoglycoprotein receptor (ASGP-R) based on the difference in their adhesion to poly[N-p-vinylbenzyl-O-beta-d-galactopyranosyl-(1-->4)-d-gluconamide] (PVLA), and examined the proliferative activity and gene expression of these fractionated hepatocytes. The results showed that approximately 0.5 to 1% of the total number of hepatocytes, which showed low adhesion to PVLA, expressed low levels of the ASGP-R, while the rest of hepatocyte population with high adhesion to PVLA expressed high levels of the ASGP-R. Interestingly hepatocytes with low ASGP-R expression levels had much higher DNA synthesizing activity (i.e., are much more proliferative) than those with high ASGP-R expression levels. Moreover, hepatocytes with low ASGP-R expression levels expressed higher levels of epidermal growth factor receptor (EGF-R), CD29 (beta1 integrin) and CD49f (alpha6 integrin) and lower levels of glutamine synthetase than those with high ASGP-R expression. These findings suggested that hepatocytes with low adhesion to PVLA due to their low ASGP-R expression could be potential candidates for progenitor-like hepatocytes due to their high proliferative capacity; hence, the low expression of the ASGP-R could be a unique marker for progenitor hepatocytes. The isolation of hepatocytes with different functional phenotypes using PVLA may provide a new research tool for a better understanding of the biology of hepatocytes and the mechanisms regulating their proliferation and differentiation in health and disease.     

Inhibition of glutamine synthetase activity by manganous ions in a cytosol extract of rat liver.

Glutamine synthetase activity in a cytosol extract of liver was inhibited non-competitively by Mn2+ ions. The apparent Ki for Mn2" in the presence of phosphate was 8 micro M. Inhibition of glutamine synthetase by intracellular Mn2+ may contribute to the very low rates of glutamine synthesis observed in perfused liver and isolated hepatocytes.