Synthesis and secretion of albumin and transferrin by foetal rat hepatocyte cultures.

Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.

The effect of cytosine arabinoside and bromodeoxyuridine on the appearance of tyrosine aminotransferase in cultured foetal hepatocytes of the rat.

1. The acquisition of dexamethasone-inducibility of tyrosine aminotransferase activity by hepatocytes cultured from 15-day-foetal rat liver is blocked in the presence of cytosine arabinoside. 2. Similar results are obtained in the presence of bormodeoxyuridine. 3. No effects on steroid-inducibility of tyrosine aminotransferase are obtained with either of the above compounds in hepatocytes cultured from 19-day-foetal liver. 4. the inhibitory effects of the agents are substantially reversed after their removal from the culture medium. 5. The effects of bromodeoxyuridine suggest that cell differentiation, with respect to tyrosine aminotransferase-inducibility, occurs in cultures of 15-day-doetal hepatocytes. 6. The effects of cytosine arabinoside suggest that such an event is dependent on mitosis.

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose.

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K(i) values being 1.37x10(-3)-1.67x10(-3)m for fructose 1-phosphate and 7.2x10(-3)-7.9x10(-3)m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K(m) values for both liver and muscle enzymes at pH7.4 and 30 degrees were 1.11x10(-4)-1.29x10(-4)m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K(m) values for glucose 6-phosphate were in the range 5.6x10(-4)-8.5x10(-4)m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.

Plasma albumin synthesis during neonatal development of the rat.

1. Liver slices were incubated with (14)C-labelled amino acids. Albumin was isolated from the slices by precipitation with specific antibody and the incorporated radioactivity measured. 2. The rate of synthesis was seen to be equal in liver slices from adult and late-stage foetal rats. 3. Synthesis was very high in the pregnant rat (three times the normal adult value) and in the 5-15-day post-natal rat (twice the normal adult value). 4. The post-natal increase may be due to the disappearance of haemopoietic tissue and its replacement by active parenchymal cells.

Development of gluconeogenesis in neonatal rat liver. Effect of premature delivery.

1. An assay method for the determination of phosphopyruvate carboxylase activity is described in which improved sensitivity is obtained by separation of the enzyme from interfering pyruvate kinase by zone sedimentation. 2. The molecular weight of rat liver phosphopyruvate carboxylase determined by zone sedimentation is about 68000. 3. Premature delivery of rat foetuses by uterine section results in the rapid appearance of phosphopyruvate carboxylase, but hexose diphosphatase and pyruvate carboxylase, already present in the foetal rat liver, are not significantly affected, and glucose 6-phosphatase activity is only slightly affected. 4. The rate of incorporation of [(14)C]pyruvate into glucose by liver slices is also greatly increased by premature delivery and there is a highly significant linear correlation between this process and the phosphopyruvate carboxylase activity.

Factors affecting the premature induction of tyrosine aminotransferase in foetal rat liver.

1. Premature delivery of foetal rats by uterine section results in the rapid appearance of tyrosine aminotransferase activity in foetal liver, after an initial lag period of 3-6hr. 2. The premature induction of activity is completely repressible by actinomycin D given soon after delivery and partially repressible by puromycin and amino acid analogues. 3. Glucagon injections into foetal rats in utero lead to production of tyrosine aminotransferase in the foetal liver, but adrenalin and nor-adrenalin are without effect. 4. Injections of glucose, galactose, fructose and mannose into prematurely delivered rats repress the development of tyrosine aminotransferase activity about 50% when they are given 2hr. after delivery, but glucose has no significant effect when injected at delivery. 5. The results are discussed in relation to current hypotheses on the role of hormones in enzyme induction in foetal development.

Gluconeogenesis from amino acids in neonatal rat liver.

1. The utilization of amino acids for gluconeogenesis by rat liver develops in postnatal life, reaching maximum activity at the fifth day. 2. The activity of aspartate transaminase shows a similar trend in postnatal development and the increased activity appears to be due to the soluble enzyme. 3. The activity of alanine transaminase is low in foetal and postnatal rat liver and increases in activity at about the twentieth day. 4. Aspartate, glutamate and alanine make a major contribution to gluconeogenesis in the postnatal rat liver.

Factors affecting the premature induction of phosphopyruvate carboxylase in neonatal rat liver.

1. Phosphopyruvate carboxylase activity rapidly appears in the liver of prematurely delivered rats and development of activity is prevented by injection of actinomycin D just before delivery. 2. The activity is considerably decreased by puromycin and amino acid analogues and thus appears to be due to enzyme synthesis. 3. Newborn or premature animals show a transient intense phase of hypoglycaemia after delivery. 4. When the hypoglycaemic phase is prevented by glucose injection little phosphopyruvate carboxylase activity appears in the liver, but galactose, mannose and fructose, which have no effect on the blood glucose concentration, also repress enzyme development. 5. Lactate, pyruvate and glycerol injections repress the premature development of phosphopyruvate carboxylase. 6. Injections of glucagon, adrenalin and noradrenalin into the rat foetus in utero result in development of phosphopyruvate carboxylase activity. 7. These findings are discussed in relation to the mechanism of initiation of enzyme synthesis in neonatal rat liver.

Plasma corticosterone concentrations in the perinatal rat.

1. Plasma corticosterone concentrations were determined in the foetal rat during the gestational period from day 18.5 to term and in postnatal rats over the first few hours after delivery. 2. The plasma corticosterone concentrations in foetal rats are as high as six times maternal values at day 19 of gestation and are approximately equal to maternal values from day 20 to term. 3. In postnatal rats the plasma corticosterone concentrations rise 3.5-fold on average within 5hr. of delivery. 4. The results are discussed in relation to the function of adrenal steroids in postnatal liver development.

Sites of synthesis of plasma proteins in the foetal rat.

1. The foetal rat of 16 or more days incorporates (14)C-labelled amino acids into all the demonstrable plasma protein fractions in vivo. 2. Slices of foetal rat liver incubated in vitro incorporate (14)C-labelled amino acids into the main plasma protein fractions, including the foetal-specific ;post-albumin'. 3. Slices of placenta are unable to incorporate (14)C-labelled amino acids into plasma proteins in vitro. 4. Liver slices from maternal rats incubated in vitro incorporate (14)C-labelled amino acids into plasma proteins. The presence of post-albumin cannot be demonstrated after incubation. 5. Liver slices from foetal rats, but not from adult rats, contain demonstrable amounts of haemoglobin into which (14)C-labelled amino acids are incorporated.

Ontogeny of the glucocorticoid receptor and its relationship to tyrosine aminotransferase induction in cultured foetal hepatocytes.

The glucocorticoid receptor activity that can be detected in the liver from 15-day foetal rats would appear to be associated with the haemopoietic cells. In hepatocytes, purified by culture for 1-2 days from 15-day foetal rats, the glucocorticoid receptor activity is low and dexamethasone does not induce the enzyme tyrosine aminotransferase. If culture is continued both receptor activity and steroid responsiveness are acquired. Cultured hepatocytes from 19-day foetal liver contain receptor from the first day of culture and, furthermore, the subsequent level of response to glucocorticoids is directly correlated with the actual receptor concentration. It would appear that the glucocorticoid receptor is not acquired by hepatocytes until after 18 days of gestation. Nevertheless, the fact that bromodeoxyuridine has no effect on the rate of accumulation of receptor in hepatocytes suggests that the differentiative event leading to the subsequent appearance of the receptor has already occurred before day 15 of gestation. However, the acquisition of the receptor would appear to be dependent on mitosis as cytosine arabinoside can inhibit the process.

Development of gluconeogenesis in neonatal rat liver. Effect of triamcinolone.

1. The normal development of the key enzymes of gluconeogenesis in rat liver, glucose 6-phosphatase, hexose diphosphatase, phosphopyruvate carboxylase and pyruvate carboxylase, was measured during the neonatal period. 2. Glucose 6-phosphatase, hexose diphosphatase and pyruvate carboxylase are all present in the late foetal liver, but all the enzymes show an increase in activity after birth. 3. Phosphopyruvate carboxylase is not present in liver extracts from foetal rats, but activity appears immediately after birth and increases rapidly over the first day and then more slowly to reach its maximum at the fourth postnatal day. 4. The fluorinated synthetic glucocorticoid, triamcinolone, was administered to foetal rats at various gestation times by intraperitoneal injection in utero and the animals were killed at intervals between 4 and 48hr. later. 5. The administration of triamcinolone results in slight depression of glucose 6-phosphatase, and a more significant depression of hexose diphosphatase to about one-half its normal activity in foetal rat liver. 6. Triamcinolone injection is without effect on pyruvate carboxylase activity and does not result in premature appearance of phosphopyruvate carboxylase in foetal rat liver. 7. Pyruvate kinase and aspartate amino-transferase activities in foetal rat liver are both depressed by triamcinolone treatment, whereas phosphofructokinase activity is elevated. 8. Tyrosine amino-transferase activity in foetal rat liver is markedly elevated in animals exposed to triamcinolone for 10hr. or more, but the effect is only observed in animals close to term. 9. The results are discussed in relation to mechanisms involved in the initial synthesis of tissue-specific enzymes in developing tissues, and it is concluded that glucocorticoids do not initiate the synthesis of the gluconeogenic enzymes.

Hepatocyte differentiation in culture. Appearance of tyrosine aminotransferase.

Liver of rat foetuses from 14 to 19 days of gestation and cultured hepatocytes derived from foetuses of 14 or 15 days gestation show a limited capacity to transaminate tyrosine. This low tyrosine transamination activity can be ascribed to aspartate aminotransferase. Definitive tyrosine aminotransferase can be demonstrated in 1-day-old cultures of hepatocytes taken from 19-day foetuses, but not from 15-day foetuses. However, after 3 days of culture hepatocytes from 15-day foetuses are able to synthesize tyrosine aminotransferase. Induction studies reveal that dexamethasone is capable of increasing tyrosine aminotransferase activity once it is detectable in culture.