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.     

Induction of glutamine synthetase in periportal hepatocytes by cocultivation with a liver epithelial cell line

Cocultures of periportal, glutamine synthetase-negative (GS-) hepatocytes with endothelial cells of human veins or epithelial cells of rat liver (clone RL-ET-14) were established for testing whether GS could be induced in the hepatocytes by interactions between the different cell types. While GS activity in endothelial cells was below detection level that of RL-ET-14 cells decreased from 62 mU/mg (24 h) to 38 mU/mg (168 h). During cocultivation with endothelial cells no change in the low GS activity could be detected. In contrast, when periportal hepatocytes were cocultured with RL-ET-14 cells, GS activity of the cocultures increased continuously from 26 mU/mg (24 h) to 56 mU/mg during cultivation for 168 h. Immunocytochemical staining of the cocultures for GS showed that this rise of GS activity was associated with an increase of GS level in the periportal hepatocytes and a decrease in the RL-ET-14 cells. Correspondingly, cultivation of periportal hepatocytes with media conditioned by the RL-ET-14 cells led to an increase in GS activity which, however, remained below that of cocultures, while conditioned medium of hepatocytes resulted in a decrease of GS activity in pure cultures of RL-ET-14 cells. "Separated" cocultures, where hepatocytes and RL-ET-14 cells reached each other only at the border of a circular area, demonstrated that induction of GS was highest in the marginal hepatocytes and lowest in those located in the center indicating that besides (a) soluble factor(s) other kinds of cell-cell interactions might be responsible for full induction of GS expression in periportal hepatocytes.     

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.     

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

Summary A 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. These investigations were supported in part by the Dutch Foundation for Medical Research FUNGO (grant 13-50-38).     

Uptake and clearance of plutonium-238 from liver cells transplanted into fat pads of F344 rats

This research is directed toward understanding the role of liver cells and the liver environment in plutonium biokinetics. Animals injected with liver cells and control animals received a single intraperitoneal injection of 37 kBq (1 microCi) 238Pu citrate and were serially sacrificed 1, 5, 10, 15, 30, 60 or 70 days later. Uptake, retention and distribution of Pu in intact liver and in liver cells growing in fat pads were determined. From these measurements, it was observed that the cells of the intact liver took up about twice as much 238Pu as liver cells transplanted into the fat pads of the same animal. The retention half-life was 8.3 days for the total activity in the liver, 20 days using tracks/cell measurements in the liver and 16 days for the tracks/cell measurements in the liver cells translocated to fat pads. When the data on tracks/cell were standardized relative to the amount of Pu present at 5 days after the injection, there was no significant difference between the retention of Pu in liver cells from intact animals and liver cells transplanted into the fat pads. About 20 per cent of the 5-day Pu liver burden in both liver cells and liver cells transplanted into fat pads was retained at 70 days. The smaller retention and clearance for liver cells in different environments indicate that uptake and clearance of Pu from the body is dependent, to a major extent, upon hepatocyte function.     

Synthesis, accumulation and turnover of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of embryonic rat hepatocytes

Glucocorticosteroid, thyroid hormones and cyclic AMP can induce the synthesis of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of hepatocytes as soon as these cells differentiate from the embryonic foregut. The low levels of both enzymes that can accumulate in such still protodifferentiated hepatocytes are due to low levels of enzyme synthesis. In cultures, the rate of synthesis of both enzymes increases continually in the presence of hormones, showing that maturation of the capacity for synthesis towards the postnatal, fully differentiated situation is occurring in these cells. The turnover rate of both enzymes in embryonic hepatocytes is lower in the presence of hormones than in the absence, but does not change during the culture period. In the presence of hormones the turnover rate is comparable to that found in adult rat liver in vivo. The development of the capacity to accumulate organ-specific enzymes in vitro (and hence the rate of enzyme synthesis) is found to be comparable to that in utero.     

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.     

Induction and differentiation of adipose-derived stem cells from human buccal fat pads into salivary gland cells

Atrophy or hypofunction of the salivary gland because of aging or disease leads to hyposalivation that affects patient quality of life by causing dry mouth, deterioration of mastication/deglutition, and poor oral hygiene status. Current therapy for atrophy or hypofunction of the salivary gland in clinical practice focuses on symptom relief using drugs and artificial saliva; therefore, there is still a need to develop new therapies. To investigate potential novel therapeutic targets, we induced the differentiation of salivary gland cells by co-culturing human adipose-derived stem cells isolated from buccal fat pads (hBFP-ASCs) with human salivary-gland-derived fibroblasts (hSG-fibros). We examined their potential for transplantation and tissue neogenesis. Following the culture of hBFP-ASCs and hSG-fibros, differentiated cells were transplanted into the submandibular glands of SCID mice, and their degree of differentiation in tissues was determined. We also examined their potential for functional tissue reconstitution using a three-dimensional (3D) culture system. Co-cultured cells expressed salivary-glandrelated markers and generated new tissues following transplantation in vivo. Moreover, cell reconstituted glandular structures in the 3D culture system. In conclusion, coculture of hSG-fibros with hBFP-ASCs led to successful differentiation into salivary gland cells that could be transplanted to generate new tissues.     

Localization of carbamoylphosphate synthetase and aspartate carbamoyltransferase in chloroplasts

The localization of carbamoylphosphate synthetase (CPSase) and aspartate carbamoyltransferase (ACTase), the first two enzymes of the pyrimidine biosynthetic pathway, in chloroplasts was investigated. In dark-grown radish (Raphanus sativus) seedlings, light induced a prominent increase in CPSase activity, but had little effect on ACTase activity. Both enzymes were found in chloroplasts isolated from radish cotyledons and leaves of spinach (Spinacia oleracea), soybean (Glycine max), and corn (Zea mays). The higher activity of ACTase relative to CPSase is discussed in relation to the instability of carbamoylphosphate, the product of the CPSase, and to the control of pyrimidine synthesis. Based on these results, the function of CPSase and ACTase in chloroplasts is discussed.     

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.     

Induction of S-adenosylmethionine synthetase isozymes in primary cultures of adult rat hepatocytes

The activities of S-adenosylmethionine synthetase isozymes were studied using adult rat hepatocytes in primary culture. Hepatocytes from adult rats were isolated and cultured for several days. The activities of the synthetase isozymes did not change during primary culture. The activity of the alpha-form increased with increasing ethionine plus adenine or methionine in the medium, and reached about 5 fold after 2 days. However, the increased activity of the beta-form showed less than twice.     

Distribution of glutamine synthetase and an inverse relationship between glutamine synthetase expression and intramuscular glutamine concentration in the horse

Glutamine plays important roles in the interorgan transport of nitrogen, carbon and energy but little is known about glutamine metabolism in the horse. In this study we determined the tissue distribution of glutamine synthetase expression in three Standardbred mares. Expression of glutamine synthetase was highest in kidney and mammary gland, and relatively high in liver and adipose tissue. Expression was lower in gluteus muscle, thymus, colon and lung, and much lower in small intestine, pancreas and uterus. The pattern of glutamine synthetase expression in the horse is similar to that of other herbivores and it is likely that skeletal muscle, liver, adipose tissue and lungs are the major sites of net glutamine synthesis in this species. Expression did not differ between adipose tissue depots but did vary between different muscles. Expression was highest in gluteus and semimembranous muscles and much lower in diaphragm and heart muscles. The concentration of intramuscular free glutamine was inversely correlated with expression of glutamine synthetase (r=-0.81, p=0.0017). The concentration of free glutamine was much higher in heart muscle (21.6+/-0.9 micromol/g wet wt) than in gluteus muscle (4.19+0.33 micromol/g wet wt), which may indicate novel functions and/or regulatory mechanisms for glutamine in the equine heart.     

Proliferation, differentiation and morphogenesis of fetal rat glandular stomach transplanted under the kidney capsule of syngeneic hosts

Undifferentiated glandular stomach tissue fragments from 16.5-day fetal rats were transplanted under the kidney capsule of syngeneic adult rats, and the proliferation, differentiation and morphogenesis of the transplanted tissues were investigated. Gastric epithelial cells began to invaginate 3–4 days after the transplantation and immature glands were formed after 1 week. During the period, there was a gradual increase in the expression of pepsinogen and cathepsin E, markers of cytodifferentiation of the stomach epithelia, both at protein and mRNA levels. Cathepsin E was weakly expressed in undifferentiated gastric epithelial cells at 16.5 days of gestation, and a higher level of the expression was observed in differentiated epithelia of the transplants. In contrast, the pepsinogen-producing cells first appeared around days 3–4 after transplantation and gradually increased in number to about 30% of the epithelial cells and became localized at the bottom of the gland. During the period of the experiment up to 1 month, the pepsinogen-producing cells were all positive for class III mucin and cathepsin E, indicating the immature character of these cells. In addition, no parietal cells were observed. When the tissue fragments were transplanted into adrenalectomized animals, the epithelial differentiation and morphogenesis was suppressed, but its proliferation was enhanced. The observed changes were reversed by hydrocortisone replacement. These results suggest that the development of the 16.5-day fetal stomach is regulated intrinsically to a certain extent by the genetic program of the cells involved and various gastric functions develop in the absence of luminal stimulation, stage-specific systemic hormonal change, neuronal regulation or other systemic influences, and that glucocorticoids modulate the developmental program of the fetal stomach tissues.