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.     

Expression patterns of mRNAs for α-fetoprotein and albumin in the developing rat: the ontogenesis of hepatocyte heterogeneity

Summary In developing and normal adult rat liver the expression patterns of the mRNAs for -fetoprotein (AFP) and albumin (ALB) were analysed byin situ hybridization using specific³⁵S-labelled complementary DNA probes. In the developing liver AFP and ALB mRNA are found from embryonic day (ED) 11 and 12, respectively, onward. At ED 20 the first signs of a zonal distribution of these mRNAs across the liver lobule can be observed, AFP mRNA concentration being higher in the pericentral area and ALB mRNA concentration higher in the periportal area. This distribution pattern of reciprocal, overlapping gradients of mRNA can be clearly recognized in the neonatal period. In the adult liver AFP mRNA can no longer be detected and similar to the neonatal situation, ALB mRNA is expressed across the entire porto-central distance decreasing in concentration going from the portal to the central area.Transient extra-hepatic expression of AFP mRNA is found in the embryonic heart and in the epithelial lining of intestine and lung furthermore, AFP and ALB mRNA are found to be transiently expressed in the developing renal tubules. Similar expression patterns have been observed for other liver-characteristic mRNAs (Moormanet al., 1990), suggesting that common regulatory factors are operative during development.     

Predominant localization of phosphoenolpyruvate carboxykinase mRNA in the periportal zone of rat liver parenchyma demonstrated by in situ hybridization

In rat liver parenchyma, expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene was studied by Northern blot analysis with a biotinylated cRNA probe and the zonal localization of PEPCK mRNA was demonstrated by in situ hybridization with a radiolabelled cRNA probe. During the feeding period at night, overall PEPCK mRNA levels were low and PEPCK mRNA was detected only in small areas of the periportal zone. At the beginning of the light period (7 am) the overall PEPCK mRNA level began increasing and the periportal areas containing PEPCK mRNA broadened. The maximum of the total abundance and of the area with high levels of PEPCK mRNA was reached at noon. Fasting for 24-72 h did not cause further significant alterations in the level or localization of PEPCK mRNA. The present data are in line with previous findings of the predominant localization of PEPCK activity and enzyme protein in periportal hepatocytes. They suggest that the heterogeneous expression of the PEPCK gene in rat liver is regulated at the pretranslational level.     

Zonal expression of hepatocytic marker enzymes during liver repopulation

Hepatocytes are metabolically specialised cells displaying distinctive gene expression patterns within the liver lobule. Here, we investigate whether pre-cultured adult rat hepatocytes adopt periportal and pericentral enzyme expression following their transplantation into the regenerating rat liver. Isolated primary rat hepatocytes, representing a mixture of both periportal and pericentral origin, lost expression of carbamoyl phosphate synthetase I (CPS I) and cytochrome P450 subtype 2B1 (CYP2B1) in culture as shown by immunofluorescence and Western blot analysis. Accordingly, urea synthesis and CYP2B1 enzyme activity decreased. Hepatocytes from DPPIV (CD26) wild type rats were cultured for 4 and 7 days, and then transplanted into the livers of CD26 deficient rats following prior treatment with retrorsine and partial hepatectomy to drive selective donor cell proliferation. CD26 positive donor cells engrafted in the periportal regions and grew in clusters expanding into the parenchyma as time proceeded. Ten weeks after transplantation, cells derived from donors surrounding the portal veins expressed CPS I, but not CYP2B1. The reverse was true for CD26 positive cells in close proximity to the central veins displaying immunoreactivity to CYP2B1, but no longer to CPS I. Hepatocytes lose their specific marker enzyme expression in culture. After transplantation, donor hepatocytes proliferate in the host parenchyma whilst acquiring the position-specific enzyme expression of the surrounding periportal and pericentral host hepatocytes. These results indicate the high degree of plasticity of gene expression in hepatocytes subjected to a change in microenvironment.     

Phosphoenolpyruvate carboxykinase activity patterns in the liver acinus of diabetic and diabetic and estrogen treated rats

Summary The acinar activity pattern of phosphoenolpyruvate carboxykinase (PEPCK) was investigated in livers of streptozotocin diabetic male and female rats and in addition in livers of diabetic males, which had undergone estrogen treatment. In all diabetic animals blood glucose levels were supranormal and liver PEPCK activity was increased. This increase in activity was greatest in estrogen treated diabetic males and lowest in diabetic females. Plasma insulin levels were reduced after the application of streptozotocin to otherwise normal male and female rats. Yet, in males treated in addition with estrogens the plasma insulin levels reached the normal range again. The PEPCK activity showed a heterotopic distribution along the acinus. The periportal to perivenous gradient was steeper in males compared to females in the untreated as well as in the diabetic state. The application of estrogens to males resulted in a further steepening of the gradient.     

Phosphoenolpyruvate carboxykinase activity patterns in the liver acinus of diabetic and diabetic and estrogen treated rats

The acinar activity pattern of phosphoenolpyruvate carboxykinase (PEPCK) was investigated in livers of streptozotocin diabetic male and female rats and in addition in livers of diabetic males, which had undergone estrogen treatment. In all diabetic animals blood glucose levels were supranormal and liver PEPCK activity was increased. This increase in activity was greatest in estrogen treated diabetic males and lowest in diabetic females. Plasma insulin levels were reduced after the application of streptozotocin to otherwise normal male and female rats. Yet, in males treated in addition with estrogens the plasma insulin levels reached the normal range again. The PEPCK activity showed a heterotopic distribution along the acinus. The periportal to perivenous gradient was steeper in males compared to females in the untreated as well as in the diabetic state. The application of estrogens to males resulted in a further steepening of the gradient.     

The effect of sex hormones on the acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in rat liver

The intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity (PEPCK) was investigated in microdissected samples of livers from normal, castrated, castrated and estradiol- or testosterone-treated, and uncastrated and testosterone- or estradiol-treated male and female rats. The total PEPCK activity showed a marked sex dependency, with 1.8 times higher activity in males. The intra-acinar distribution profiles were also sex-dependent. The periportal-to-perivenous gradient was steeper in males. Castration resulted in an approximation of PEPCK activity and its acinar distribution pattern between the sexes due to a reduction in males and an increase in females. Estrogen treatment of castrated males had no further effect on PEPCK activity and its acinar gradient, whereas in ovariectomized animals the activity was reduced to levels near normal. Testosterone treatment of castrated male or female animals led to a marked increase in enzyme activity with a concomitant steepening of the acinar gradient. Administration of estradiol to normal male rats also led to a reduction in activity, together with a change in the acinar activity gradient. Testosterone treatment of normal females resulted in an induction of PEPCK activity which was most prominent in the periportal zone. The most drastic changes were observed in the perivenous zones. In all experiments a periportal-to-perivenous activity gradient persisted thus marking the periportal zone as the area with highest gluconeogenic capacity.     

Periportal zonation of the cytosolic acetyl-CoA synthetase of male rat liver.

Several important metabolic functions of the mammalian liver have been shown to be located in zones with respect to the complex microcirculation of the organ. The zonal distribution of the cytosolic component of the acetyl-CoA synthetase activity has been investigated using the dual-digitonin-pulse-perfusion technique, which allows highly zone-selective sampling of cytosol from the periportal and perivenous zone of rat liver. Approximately 80% of the cytosolic enzymes are eluted from the hepatocytes in the periportal and perivenous sub-zones affected by digitonin, while less than 1% of the glutamate dehydrogenase activity (a marker enzyme of the mitochondrial compartment) is eluted. A twofold higher activity of the cytosolic form of acetyl-CoA synthetase is found in the periportal zone compared to the perivenous zone in fed male rats. Following a fasting/refeeding transition, this activity gradient is abolished in a manner similar to that observed for the enzyme acetyl-CoA carboxylase. Since the latter enzyme is utilizing the product of acetyl-CoA synthetase, acetyl-CoA, the similarity in the observed regulation suggests a functional coupling between cytosolic acetate activation and fatty-acid synthesis.     

Effects of starvation and refeeding a high carbohydrate diet on the intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in the liver of male and female rats

Phosphoenolpyruvate carboxykinase activity in rat liver was shown to be heterotopically distributed within the acinus under varying feeding conditions. Highest values of PEPCK activity were found in the periportal zone of the acinus from where it decreased continuously towards the perivenous zone. 84 h of starvation resulted in an increase of activity, which was most prominent in the perivenous zone, but nevertheless resulted in a steeper gradient. Refeeding of starved rats with a high carbohydrate diet for 6 nights led to a decrease in PEPCK activity which was most prominent in the periportal zone, but almost negligible in the perivenous zone, resulting in a further change in the activity gradient. Sex-dependent differences for total PEPCK activity were found i) in controls, where the activity was lower in females, ii) after starvation, where the induction was much higher in females, and iii) after refeeding of starved rats, where the activity in females remained higher compared to that of the controls. Differences in the intra-acinar localization of the activity in dependence of the sex were registrated in the control group and in starved rats. Livers from female rats contained a higher periportal/perivenous ratio compared to males. In starved and starved and refed animals the periportal/perivenous ratios were almost the same in both sexes.     

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.     

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 dexamethasone-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.     

Effects of starvation and refeeding a high carbohydrate diet on the intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in the liver of male and female rats

Summary Phosphoenolpyruvate carboxykinase activity in rat liver was shown to be heterotopically distributed within the acinus under varying feeding conditions. Highest values of PEPCK activity were found in the periportal zone of the acinus from where it decreased continuously towards the perivenous zone. 84 h of starvation resulted in an increase of activity, which was most prominent in the perivenous zone, but nevertheless resulted in a steeper gradient. Refeeding of starved rats with a high carbohydrate diet for 6 nights led to a decrease in PEPCK activity which was most prominent in the periportal zone, but almost negligible in the perivenous zone, resulting in a further change in the activity gradient.Sex-dependent differences for total PEPCK activity were found i) in controls, where the activity was lower in females, ii) after starvation, where the induction was much higher in females, and iii) after refeeding of starved rats, where the activity in females remained higher compared to that of the controls. Differences in the intra-acinar localization of the activity in dependence of the sex were registrated in the control group and in starved rats. Livers from female rats contained a higher periportal/perivenous ratio compared to males. In starved and starved and refed animals the periportal/perivenous ratios were almost the same in both sexes.     

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.     

Air-breathing catfish, Clarias batrachus upregulates glutamine synthetase and carbamyl phosphate synthetase III during exposure to high external ammonia

We assessed the possible upregulation of glutamine synthetase (GS) and typical 'fish type' carbamyl phosphate synthetase III (CPS III) in detoxification of ammonia in different tissues of the walking catfish (Clarias batrachus) during exposure to 25 mM NH(4)Cl for 7 days. Exogenous ammonia led to an increase in ammonia and urea concentrations in different tissues. The results revealed the presence of relatively high levels of GS activity in the brain, liver and kidney, unexpectedly, also in the muscle, and even higher levels in the intestine and stomach. Exposure to high external ammonia (HEA) caused significant increase of activities of GS, CPS III and CPS I-like enzymes, accompanied with the upregulation of GS and CPS III enzyme proteins in different tissues. Exposure to HEA also led to a sharp rise of plasma cortisol level, suggesting being one of the primary causes of upregulation of GS and CPS III enzymes activity. Liver perfusion experiments further revealed that exposure to HEA enhances the capacity of trapping ammonia to glutamine and urea by the liver of walking catfish. These results suggest that the upregulation of GS and CPS III activity in walking catfish during exposure to HEA plays critical roles to ameliorate the toxic ammonia to glutamine, and also to urea via the induced ornithine-urea cycle possibly through the involvement of cortisol.     

The development of the acinar heterotopic pattern of phosphoenolpyruvate carboxykinase activity in the newborn rat

The postnatal appearance of phosphoenolpyruvate carboxykinase activity (PEPCK) and acinar heterotopy was investigated in newborn rats aged 2 h, 12 h, 24 h and 3 days, as well as in juvenile rats aged 25 days. The livers showed an almost homogeneous distribution of activity along the sinusoidal length at the beginning of extrauterine life where energy needs are greatest. Compared to rats aged 2 h, the PEPCK activity was higher in the livers from rats aged 12 h. The increase in activity was most pronounced in the intermediary zone. After 24 h of extrauterine life the activity decreased again creating a homogeneous acinar activity pattern. By day 3 activity had increased in the periportal zone, while decreasing in the perivenous zone, resulting in a periportal to perivenous gradient. By day 25 total activity had reached highest values both in males and females, due to a relatively high perivenous activity. The more prominent acinar gradient corresponded approximately to the one seen in adult animals.     

The development of the acinar heterotopic pattern of phosphoenolpyruvate carboxykinase activity in the newborn rat

Summary The postnatal appearance of phosphoenolpyruvate carboxykinase activity (PEPCK) and acinar heterotopy was investigated in newborn rats aged 2 h, 12 h, 24 h and 3 days, as well as in juvenile rats aged 25 days. The livers showed an almost homogeneous distribution of activity along the sinusoidal length at the beginning of extrauterine life where energy needs are greatest. Compared to rats aged 2 h, the PEPCK activity was higher in the livers from rats aged 12 h. The increase in activity was most pronounced in the intermediary zone. After 24 h of extrauterine life the activity decreased again creating a homogeneous acinar activity pattern. By day 3 activity had increased in the periportal zone, while decreasing in the perivenous zone, resulting in a periportal to perivenous gradient. By day 25 total activity had reached highest values both in males and females, due to a relatively high perivenous activity. The more prominent acinar gradient corresponded approximately to the one seen in adult animals.     

Sex differences of the influence of T3 on the topical distribution of phosphoenolpyruvate carboxykinase activity in the liver acinus

Summary Phosphoenolpyruvate carboxykinase (PEPCK) activity was investigated in livers of triiodothyronine (T₃) treated male and female rats with special regard to its intraacinar localization. In untreated controls of both, male and female rats, the activity was heterotopically distributed within the acinus with highest values in the periportal zone, and with lowest values in the perivenous zone. This periportal to perivenous activity gradient revealed to be under the influence of T₃. Application of T₃ resulted in a relative increase of PEPCK activity which was much greater in the livers of females than in males. The extent of T₃-induced augmentation of PEPCK activity was dependent on the intraacinar position. In both sexes greatest relative activation was found in the perivenous zone. In female animals, the perivenous activity of T₃ treated livers was comparable to that observed in the periportal zone of controls.     

Gluconeogenic-glycolytic capacities and metabolic zonation in liver of rats with streptozotocin, non-ketotic as compared to alloxan, ketotic diabetes

Activities (mumol X min-1 X g liver) and zonal distributions of key enzymes of carbohydrate metabolism were studied in livers of streptozotocin-diabetic rats and compared to the values in alloxan-diabetes. Streptozotocin led to a non-ketotic diabetes with blood glucose being increased by more than fivefold but ketone bodies being in the normal range, while alloxan produced a ketotic diabetes with blood glucose, acetoacetate and beta-hydroxybutyrate being elevated by more than fivefold. Portal insulin was decreased to about 20% in streptozotocin- and more drastically to about 7% in alloxan-diabetes. Conversely, portal glucagon was increased in the two states to about 250% and 180%, respectively. The glucogenic key enzyme phosphoenolpyruvate carboxykinase (PEPCK) was enhanced in streptozotocin- and alloxan-diabetes to over 300%, while the glycolytic pyruvate kinase L (PKL) was lowered to 65% and 80%, respectively. The normal periportal to perivenous gradient of PEPCK of about 3:1, as measured in microdissected tissue samples, was maintained with elevated activities in the two zones. The normal periportal to perivenous gradient of PKL of 1:1.7 was diminished with lowered activities in the two zones. The glucogenic glucose-6-phosphatase (G6Pase) was increased in streptozotocin- and alloxan-diabetes to 130% and 140%, respectively, while the glucose utilizing glucokinase (GK) was decreased to 60% and 50%, respectively. The normal periportal to perivenous gradient of G6Pase, demonstrated histochemically, remained unaffected. Carnitine palmitoyltransferase (CPT) was increased to over 190% and acetyl-CoA carboxylase (ACC) was decreased to 60% in streptozotocin, non-ketotic diabetes, while the two enzymes were altered more drastically to 400% and 50%, respectively, in alloxan, ketotic diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)