Hepatic enzymic zonation: a reevaluation of the concept of the liver acinus.

The distribution pattern of a periportal enzyme (carbamoylphosphate synthetase) and a pericentral enzyme (glutamine synthetase) in human and rat liver has provided an objective parameter to delineate the zonal boundaries of the liver acinus. On sections, the pericental zone (zone 3) is circular and discrete rather than star-like and reticular, as predicted by the acinar concept, whereas the periportal zone (zone 1) is reticular, i.e. contiguous between adjacent acini rather than discrete. Three-dimensionally, the composite of pericentral zones (the pericentral compartment) follows the branching pattern of the terminal hepatic (central) vein, whereas the composite of periportal zones (the periportal compartment) envelops the pericentral compartment as a three-dimensional network (reticulum). This modified concept that is based upon the three-dimensional distribution of hepatocyte-specific enzymes is supported by data from the literature regarding the three-dimensional angioarchitecture of the liver, the perfusion pattern of the liver and the three-dimensional pattern of tissue oxygenation. Hence, a unified concept of the liver architecture that is based upon the observed distribution pattern of blood flow, of gene expression and of metabolism can be established.

The gamma-glutamyltransferase/glutamine synthetase activity ratio. A powerful marker for the acinar origin of hepatocytes.

The activity of glutamine synthetase (GS) in hepatocytes isolated by digitonin-collagenase perfusion from the perivenous region was more than 10-times higher than in cells isolated from the periportal region. This distribution was confirmed by immunohistochemical staining for GS of cells separated from either region. In contrast, in periportal hepatocytes, the activity of gamma-glutamyltransferase (GGT) was 3-4 times as high as in perivenous hepatocytes. This acinar distribution was also confirmed histochemically. The striking reciprocal acinar distribution of these two enzymes, now observed by direct biochemical analysis of selectively isolated hepatocytes, confirms the earlier qualitative differences observed by histochemistry and immunohistochemistry. The GGT/GS ratio seems to serve as a powerful marker of the acinar origin of isolated hepatocyte populations. Preliminary data describing glutamine synthetase activity in plasma of some subjects with suspected liver dysfunction suggests this enzyme as a marker for pericentral damage.

Gene expression in derivatives of embryonic foregut during prenatal development of the rat.

Proteins characteristic for the adult cellular phenotype, i.e., carbamoylphosphate synthetase (CPS) for liver and small intestine, arginase for liver, glutamate dehydrogenase (GLDH) for pancreas, liver, and small intestine, and amylase for pancreas were studied immunohistochemically in rat embryos and fetuses. At distinct developmental stages, subsets of enzymes appear synchronously in the foregut derivatives, suggesting that gene expression in the different organs is regulated by common factors. In contrast to the long-held opinion that fetal hepatocytes are a homogeneous cell population, it is shown that arginase and CPS are heterogeneously distributed between ED 16 and ED 20. This heterogeneity is related to the vascular architecture of the liver and disappears perinatally as the result of strong stimulation of enzyme synthesis. In addition, an intercellular heterogeneity in CPS content that is not related to the vasculature is observed between ED 14 and ED 20. This "random" heterogeneity reflects temporal differences in the onset of CPS accumulation in individual cells.

Immunohistochemical localization of glutamate dehydrogenase in rat liver: plasticity of distribution during development and with hormone treatment.

In adult rat liver, glutamate dehydrogenase is present in high concentrations around the terminal portal (zone 1) and hepatic (zone 3) veins, whereas its concentration is low in the intermediate zone. Although the size and staining intensity of the periportal glutamate dehydrogenase-positive compartment are less than those of the pericentral compartment, it can expand under appropriate endocrine conditions, leading to a homogeneous distribution. At birth, glutamate dehydrogenase is also homogeneously distributed. Glutamate dehydrogenase disappears from the periportal compartment during the first postnatal week and reappears in that compartment after weaning. These observations indicate an independent regulation of glutamate dehydrogenase levels in the periportal and pericentral zone. The size of the periportal glutamate dehydrogenase-containing zone is appreciably smaller than that of carbamoylphosphate synthetase, whereas the pericentral glutamate dehydrogenase-containing zone is appreciably larger than that of glutamine synthetase. The heterogeneous distribution of glutamate dehydrogenase suggests the possibility that, under normal conditions, deamination of glutamate prevails in the periportal compartment and amination of glutamate in the pericentral compartment.

Reciprocal regulation of glutamine synthetase and carbamoylphosphate synthetase levels in rat liver.

In glucocorticosteroid-treated diabetic rats, glutamine synthetase enzyme levels in the liver are decreased 3-fold, whereas carbamoylphosphate synthetase enzyme levels are increased 2.3-fold. In addition, immunohistochemistry shows that under these conditions the distribution of carbamoylphosphate synthetase is expanded over the entire liver acinus, whereas that of glutamine synthetase is reduced to very few cells bordering the central (terminal hepatic) veins. Using a newly isolated cDNA complementary to rat liver glutamine synthetase mRNA, we show that this regulation is primarily effected at a pretranslational level. (For data on carbamoylphosphate synthetase mRNA levels, see De Groot et al. (1986) Biochim. Biophys. Acta 866, 61-67). Furthermore, hybridization studies show stimulatory effects of both glucocorticosteroids and thyroid hormone on the glutamine synthetase mRNA level. Attempts to localize glutamine synthetase mRNA within the liver acinus by selective destruction of the pericentral zone failed because of generally low levels of liver mRNAs after CCl4 poisoning. In contrast to the situation after birth, significantly higher glutamine synthetase mRNA/enzyme activity ratios in fetal rat liver point to the presence of additional post-transcriptional control mechanisms before birth. These findings complement similar observations on carbamoylphosphate synthetase gene expression (De Groot et al. (1986) Biochim. Biophys. Acta 866, 61-67).

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.

Heterogeneous distribution of glutamine synthetase during rat liver development.

Two days before birth, immunohistochemical detection of glutamine synthetase already reveals a heterogeneous distribution pattern related to the vascular architecture of the liver. Only a small number of hepatocytes in the vicinity of the efferent venules show relatively high staining intensity. Before that age, only megakaryocytes show intense staining, while liver parenchyma is only faintly stained. The developmental profile of glutamine synthetase activity shows two periods of increasing enzyme activity: one in the perinatal period and one in the second and third postnatal week. Both periods are correlated with high levels of circulating corticosteroid hormones. Although the relative number of intensely stained hepatocytes increases during the first rise in enzyme activity, the second rise is correlated with a decreasing number of glutamine synthetase-positive hepatocytes which, however, show a considerable increase in staining intensity. Carbamoylphosphate synthetase shows a homogeneous distribution pattern in the perinatal period. Conditions that lead during development to a relatively high level of glutamine synthetase expression in the pericentral compartment apparently originate before the appearance of conditions that lead to a relatively high level of carbamoylphosphate synthetase gene expression in the periportal compartment. Our results indicate that downstream localization of glutamine synthetase in liver acinus is essential from the perinatal period onwards, whereas reciprocal distribution of glutamine synthetase and carbamoylphosphate synthetase gene expression (that is found in adult rat liver) is not.

Development of the heterogeneous distribution of carbamoyl-phosphate synthetase (ammonia) in rat-liver parenchyma during postnatal development.

Carbamoyl-phosphate synthetase (ammonia) is homogeneously distributed in rat-liver parenchyma at birth, as demonstrated by immunohistochemistry. A heterogeneous distribution can first be demonstrated at 6 days post partum, but can be masked by use of a too sensitive detection system. This heterogeneity is established by a decrease in enzyme content around the hepatic venules and a considerable increase in enzyme content in the remaining parenchyma. The perivenous decrease in enzyme content does not occur in all hepatocytes synchronously. The adult type of heterogeneity is characterized by a perivenous layer, only two to three cells thick, in which carbamoyl-phosphate synthetase can no longer be detected, irrespective of the sensitivity of the assay used. This situation is fully established at the age of two months.

Immunohistochemical localization of carbamoyl-phosphate synthetase (ammonia) in adult rat liver; evidence for a heterogeneous distribution.

Different fixation media have been compared in order to find one that preserves the histological structure of rat liver and allows unambiguous immunohistochemical detection of carbamoyl-phosphate synthetase (ammonia). Fixation of rat liver in a mixture of methanol, acetone, and water yields the most intense immunohistochemical staining. Using a specific antiserum raised against rat liver carbamoyl-phosphate synthetase, less than 1% of the enzyme protein is extractable after this fixation procedure, and the histological structure is similar to that after fixation in Bouin's fixative. Specific immunohistochemical staining is localized exclusively in the cytoplasm of the parenchymal cells; its granular distribution is in accordance with the mitochondrial localization of carbamoyl-phosphate synthetase. Immunohistochemical staining shows a heterogeneous distribution within the liver acinus. Staining is most intense around the portal venules, decreases slowly toward the hepatic venules and is, after an abrupt decrease, virtually absent in a limited area surrounding these venules. The possible significance of the heterogeneous distribution of carbamoyl-phosphate synthetase for ammonia metabolism is discussed.