Synthesis of human gamma-glutamyl transpeptidase (GGT) during the fetal development of liver

We have determined expression of human GGT gene encoding gamma-glutamyl transpeptidase (GGT) during fetal development of liver using the Northern-blot analysis with a cloned human GGT cDNA and immunohistochemistry with a monoclonal antibody. GGT mRNA could be detected as early as the 12th week of gestation. It then increased gradually to a peak of approx. threefold the amount at week 12, at week 40, just before birth. The size of the mRNA in the fetal liver was 2.7 kb and mRNA of the same size was detected both in the human fetal kidney and human hepatocellular carcinoma as well as normal adult liver. Immunohistochemical analyses show that GGT increased as the fetal liver developed in parallel with the increase in mRNA. Histochemically, GGT was shown to be located in the wall of bile canaliculi when synthesis was low in early development, but to be distributed, in addition, all over the cell membrane of the fetal hepatocytes when synthesis was high at the later stage of development.     

Ontogeny of microsomal activities of triacylglycerol synthesis in guinea pig liver

Because the onset of triacylglycerol-rich lipoprotein synthesis occurs in guinea pig liver during fetal life, we investigated the microsomal enzyme activities of triacylglycerol synthesis in fetal and postnatal guinea pig liver. Hepatic monoacylglycerol acyltransferase specific and total microsomal activities peaked by the 50th day of gestation and declined rapidly after birth to levels that were virtually unmeasurable in the adult. Peak fetal specific activity was more than 75-fold higher than observed in the adult. The specific activities of fatty acid CoA ligase and lysophosphatidic acid acyltransferase increased 2- to 3-fold before birth; lysophosphatidic acid acyltransferase increased a further 2.6-fold during the first week of life. Specific activities of phosphatidic acid phosphatase, microsomal glycerophosphate acyltransferase, and diacylglycerol acyltransferase varied minimally over the time course investigated. These data demonstrate that selective changes occur in guinea pig hepatic microsomal activities of triacylglycerol synthesis before birth. Because of an approximate 11-fold increase in hepatic microsomal protein between birth and the adult, however, major increases in total microsomal activity of all the triacylglycerol synthetic activities occurred after birth. The pattern of monoacylglycerol acyltransferase specific and total microsomal activities differs from that of the rat in occurring primarily during the last third of gestation instead of during the suckling period. This pattern provides evidence that hepatic monoacylglycerol acyltransferase activity probably does not function to acylate 2-monoacylglycerols derived from partial hydrolysis of diet-derived triacylglycerol.     

Activity of alcohol dehydrogenase and acetaldehyde dehydrogenases in the liver and placenta during the development of the rat

The ontogenetic development of the enzymes alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenases (ALDH I and II) was followed in rats. ADH could be detected just before birth and increased gradually to reach 82% of adult values at 47 days. ALDH I and II were present from day 15 of gestation, increased rapidly at birth, and reached 80-90% adult values at 47 days. The ratio between ALDH and ADH activities decreased gradually during ontogenesis. The relative subcellular distribution of all enzymes was identical before birth, 7 days after birth and in adults. The placental activities of ADH and ALDH I and II were studied at 15 and 20 days of pregnancy. ADH could not be detected in placentas. Low activities of ALDH I and II were present in placentas studied at 15 days of gestation, and still lower activities were found in placenta at 20 days.     

Investigation of thyroxine monodeiodinase activity in the liver, kidney and brown adipose tissue of foetal and neonatal rabbit

The maturation of the 5'- and 5-monodeiodinase system in liver, kidney and brown adipose tissue of rabbits, during the foetal period (from 21 days of gestation to birth) and the neonatal period (from birth to 3 weeks of life) was studied. A sudden increase of 5'- and 5-monodeiodinase activity in liver and kidney 3 days before birth was observed, falling to a nadir at day 3 after birth. Foetal and neonatal serum T4, T3 and rT3 concentration were very low and rose progressively with age, reaching adult values at about day 21. In the foetal brown adipose tissue high 5'-monodeiodinase and low 5-monodeiodinase activity was found. The 5'-monodeiodinase decreased during the first days of life whereas the 5-monodeiodinase activity remained at a low stable level until day 3 when the activities of both enzymes increased. The increase of conversion rate of T4 to T3 and rT3 in liver and kidney well correlate with the triiodothyronines concentration in serum from day 3 after birth.     

Developmental Changes in Rat Liver Xanthine Oxidase(s) and Its Intracellular Distribution

Developmental change and subcellular distribution of xanthine oxidase in the rat liver were examined.

The specific activity of the fetal liver xanthine oxidase increased sharply to the levels of the adult liver on the day of the birth. After birth, the activity dropped rapidly and on the 14th day after birth it was about 1/4 of adult level. Then the activity was regained and around 28th day after birth it was about the same as in adult level.

In the livers from 80 days old rats, about 60% of total xanthine oxidase activity was found in soluble fraction and the rest was distributed among particulate fractions including microsomal, lysosomal, mitochondrial and nuclear fractions.

In contrast to the adult livers 80% of total xanthine oxidase activity in fetal liver was found to be in particulate fractions.

From kinetic studies of xanthine oxidases in particulate and soluble fractions it was suggested that xanthine oxidase in soluble fraction and xanthine oxidase in particulate fraction might be different in their natures of protein molecule.     

Prenatal and postnatal changes in the content and species of ferritin in rat liver

The iron and ferritin content of rat liver and the species of ferritin present were examined from 4 days before to 3 weeks after birth. 1. Total iron and ferritin iron accumulated rapidly during the last days of gestation and from the second postnatal day underwent a steady depletion. 2. The amount of iron deposited before birth in the liver of each pup varied inversely with litter size and could be increased moderately by injection of iron into the mother before mating. 3. Intraperitoneal injection of iron 1 day after birth doubled the concentration of total iron, ferritin iron and ferritin protein in the liver over the next 24h, but at 3 weeks after birth it raised the very low concentrations of iron and ferritin severalfold. 4. As shown by electrophoretic migration, ferritin and dissociated ferritin subunits prepared from the livers of rats from 4 days before to 3 weeks after birth differed from those of adult liver ferritin and were indistinguishable from those of adult kidney and spleen ferritin. Treatment with iron at 3 weeks of age induced formation of a ferritin with electrophoretic properties resembling those of adult liver. It is concluded that iron given at this stage of development may activate the genetic cistron for adult liver ferritin.     

Rat liver and intestinal mucosa differ in the developmental pattern and hormonal regulation of carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase gene expression

cDNA probes were employed to measure levels of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyltransferase (OCT) mRNAs in fetal and neonatal livers and intestines. In the fetal liver, significant levels of OCT mRNA were present at 15-days gestation while CPS mRNA could not be detected until day 17 of fetal development. Apart from a small decline just after birth, amounts of both mRNAs increased steadily to reach adult levels in postnatal life. In contrast to the situation in liver, CPS and OCT mRNA levels in the fetal intestine rose rapidly to peak at day 21 of gestation and then declined steadily in the first seven days after birth. Using the methyl-sensitive restriction isoschizomeric pair, MspI/HpaII, the 5' ends of both the CPS and OCT genes were shown to undergo demethylation during development. In the case of the OCT gene, however, the hypomethylation characteristic of the adult liver and intestinal mucosa was not observed in the 15-day-old fetal liver, where significant levels of gene expression had already been established. Levels of CPS and OCT mRNA in livers of adults responded to glucagon in normal animals (1.5-fold and 2.2-fold increases, respectively) and to dexamethasone in experimentally induced diabetic animals (3-fold increase in CPS mRNA with no change in OCT mRNA). These treatments were all without effect on the levels of CPS and OCT mRNA in intestinal mucosa.     

The appearance, properties, and functions of gluconeogenic enzymes in the liver and kidney of the guinea pig during fetal and early neonatal development.

The changes in the activity and properties of the four gluconeogenic enzymes have been followed during development of the guinea pig. Pyruvate carboxylase was almost exclusively mitochondrial and kinetically identical to the adult liver enzyme and did not appear in significant activity until after day 50 when it rose to values several times higher than those in the adult liver, then fell after birth. Little activity was detected in the fetal kidney. Phosphoenolpyruvate carboxylase appeared in the fetal liver from day 30 on, both in the mitochondrial and cytoplasmic fractions. The cytoplasmic enzyme was kinetically and chromatographically identical to the mitochondrial enzyme of the fetal and maternal liver. After birth the activity of the cytoplasmic enzyme increased and that of the particulate enzyme fell. Fetal kidney activity appeared several days before birth. Fructose 1,6-diphosphatase and glucose 6-phosphatase appeared in the fetal liver and kidney after day 40; the former showed no postnatal change while the latter rose 10-fold after birth. Fetal liver fructose 1,6-diphosphatase was more sensitive to AMP and fructose 1,6-diphosphate inhibition but was chromatographically indistinguishable from the maternal liver enzyme. Despite the presence of the gluconeogenic enzymes, gluconeogenesis and glyconeogenesis were not detected in the fetal liver until 7–9 days before birth. While the synthesis of glyceride-glycerol from 3-carbon compounds was detected from 35–40 days onwards and some of the gluconeogenic enzymes participate in that pathway, gluconeogenesis was not detected in the fetal kidney.     

Developmental changes in rat hepatic casein kinases 1 and 2

Cytosolic histone kinase and casein kinase activities varied considerably in the late fetal and postnatal periods of liver development. Both activities showed a maximum at day 21 of gestation and decreased at birth to values close to those of adult rats. The changes in total casein kinase activity were due to variations of casein kinase 1 and casein kinase 2. Similarly the activities of both the cyclic-AMP-dependent protein (histone) kinase and the cyclic-AMP-independent histone kinase varied during development. Besides the changes in total activity, the affinity of casein kinases 1 and 2 for casein also varied in fetal and postnatal development. The Km values of casein kinase 2 increased from day 18, reached a maximum at day 20 of gestation and then started to decrease until one day after birth. In contrast the Km values of casein kinase 1 decreased from day 18, reached its lowest value at day 21 of gestation and attained values similar to those in the adult at the day of birth. Changes in this parameter were also observed when insulin (3 IU/kg) was administered by intraperitoneal injection to one-day-old rats. The Km values of casein kinase 1 decreased while those of casein kinase 2 increased after administration of this hormone. On the other hand, the Km values for ATP of casein kinases 1 and 2 as well as their apparent molecular masses and sensitivity to heparin and GTP did not significantly change during ontogeny of rat liver.     

Perinatal development of hepatic cholesterol synthesis in the rat

Rates of cholesterol synthesis and HMG CoA reductase activity in rat liver, have been reported to be high before and low after birth. The timing of the decline in perinatal rates of cholesterol synthesis, however, is uncertain. These studies, therefore, determined in vivo rates of cholesterol synthesis using [3H]water and hepatic reductase activity in vitro in perinatal rats. The lipid composition of the plasma, liver and its microsomal subfraction were also determined. Reductase activity increased during late gestation, remained high immediately after birth, then decreased with the commencement of suckling. Rates of cholesterol synthesis increased from gestation day 18 to 20, but in contrast to reductase activity, decreased on the day before birth. Plasma cholesterol and triacylglycerol levels increased to gestation day 19, then decreased to term. By the 6th h after birth, plasma and liver cholesterol and triacylglycerol levels had increased markedly. By 48 h after birth, the high hepatic cholesterol content was associated with an increase in the cholesteryl ester fraction. The microsomal cholesterol/phospholipid molar ratio decreased from gestation day 16 until 12 h after birth, then increased markedly from 36 to 48 h. There was an apparent inverse relationship between the change in microsomal cholesterol/phospholipid molar ratio and the fatty acid unsaturation index from gestation day 16 to 36 h after birth. The results suggest that in late gestation and before suckling, the low in vivo rate of hepatic cholesterol synthesis may not be due to low activity of HMG CoA reductase.     

Rat liver glycogen metabolism in the perinatal period

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen sythase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximun on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose of AMP on the respective phosphatases was considered. It was found that the Km of phosphatase for glucose in the prenatal period was 5–7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6–15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time.Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver.It is suggested that the rapid changes observed immediately after birth are due to an effect of hypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

Rat liver glycogen metabolism in the perinatal period.

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen synthase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximum on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose or AMP on the respective phosphatases was considered. It was found that the Km of phosphorylase phosphatase for glucose in the prenatal period was 5--7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6--15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time. Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver. It is suggested that the rapid changes observed immediately after birth are due to an effect of gypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

Levels of metallothionein messenger RNA in foetal, neonatal and maternal rat liver

We have used translation in vitro of hepatic polyadenylated RNA to characterize the levels of metallothionein mRNA in foetal, neonatal, pregnant and nulliparous rats. The translation products of foetal hepatic metallothionein mRNA increased relative to other mRNA translation products from day 18 of gestation to birth and attained a maximum, maintained throughout suckling, which is tenfold above 17-day foetal hepatic levels and fourfold above adult levels. Maternal liver metallothionein mRNA decreased fivefold between 17 days and 20 days of gestation, rose sharply immediately before birth, and was low throughout lactation.     

Ontogeny of two vitamin A-metabolizing enzymes and two retinol-binding proteins present in the small intestine of the rat.

The patterns of expression of cellular retinol-binding protein (CRBP), cellular retinol-binding protein, type two [CRBP(II)], lecithin: retinol acyltransferase (LRAT), and microsomal retinal reductase were examined for rat small intestine during the perinatal period. CRBP was present (15 pmole per mg soluble protein) at the earliest time examined, the 16th day of gestation, declining by 70% by birth, maintained to adulthood. In contrast, CRBP(II) appeared 2-3 days before birth, rising to its highest level (500 pmole per mg soluble protein) by day 3 after birth, then declining by 50% during the late suckling period to the adult level. Immunohistochemistry revealed that CRBP(II) initially appeared in the epithelial cell layer in a patchy manner, resolving by birth into an even staining of all villus-associated enterocytes. In contrast, CRBP was evenly expressed in the epithelial cell layer at day 17/18 but was absent by birth. Intestinal LRAT activity increased rapidly in the 2 days prior to birth, then declined at weaning to the adult level. Microsomal retinal reductase was measurable in the intestine at birth, but not detected during the early suckling period, reappearing at day 21. Considerable increase was then observed coincident with weaning, when carotenes, from which retinal is derived, became an important source of vitamin A. The pattern of appearance of these elements appears to prepare the intestine for the necessary processing of vitamin A required after birth.     

Developmental changes in alcohol-dehydrogenase activity in rat and guinea-pig liver

1. Alcohol-dehydrogenase activity is first detectable in the rat foetus on about the eighteenth day of gestation, after which time it increases to about 25% of the adult activity at birth. Adult activity is reached at about 18 days after birth. The ethanol-oxidizing capacity of liver slices from rats correlates well with the increase of the enzyme activity in vitro. 2. In the guinea pig there is a steady linear increase from about 17 days before term to 5 days after birth. Adult activity is reached between the sixth and eighth postnatal day. 3. Some kinetic properties of liver alcohol dehydrogenase are very similar in newborn and adult rats. 4. Administration of ethanol to pregnant rats during the latter half of gestation had no effect on alcohol-dehydrogenase activity in the liver of the newborn offspring. Intraperitoneal injections of ethanol to newborn and young rats had no effect on the alcohol-dehydrogenase activity of the livers. 5. Intraperitoneal injections of hydrocortisone and triamcinolone to newborn and adult non-adrenalectomized rats had no significant effect on the increase of the alcohol-dehydrogenase activity as studied up to 4 days after the injection.     

Hexokinase isoenzymes in tissues of the adult and developing guinea pig

Changes in the activities and isoenzyme distribution of hexokinase were determined in a number of tissues during the development of the guinea pig. The total activity in the fetal liver showed a large fall during the second half of gestation to reach adult values by term. With normal diet the fetal, neonatal, and adult livers had isoenzymes I and III but little or no detectable IV (glucokinase). The fetal liver had predominantly type I, but the proportion of type III increased during development. The kinetics of the guinea pig isoenzymes were similar to those reported for the rat. Two additional isoenzymes with mobility between I and II were detected in the fetal liver and blood. They appear to have kinetic properties similar to type I. Detectable liver glucokinase activity was induced by glucose administration to adult guinea pigs. The total activity in kidney, brain and skeletal muscle showed a postnatal rise while in the fetal heart it was high and declined after birth. These tissues contained predominantly type I with varying proportions of type III hexokinase. The ratio of particulate-bound to soluble hexokinase varied from tissue to tissue. All except the liver showed a significant increase in binding after birth. The changes are discussed in relation to the control of glucose utilization in the fetal and neonatal periods.     

Perinatal development of glucose-6-phosphatase and fructose-1,6-diphosphatase activities in pig liver

The activity of glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphosphatase (FDPase) was determined in the homogenate of the liver of 69 pig fetuses during the last third of gestation (80th to 114th day), 47 piglets from birth to 4 weeks old (suckling period) and to slaughter pigs. G6Pase is evident in fetal liver at an early date and raises steadily during gestation. In newborn piglets, the enzyme activity increases rapidly during the first hours of life and remains at this high level during the first week of life. Afterwards the enzyme activity returns to birth level, which exists also in pigs at slaughtering. The activity of FDPase is constant during the fetal period. After birth enzyme activity rises at a lower rate than the G6Pase during the first week of life. This level remains constant during the suckling period and increases thereafter until the time of slaughtering of pigs. The role of hormones in the perinatal development of these enzymes is described. Probably, thyroxine causes the prenatal increase of the activity of both the enzymes. The rapid postnatal rise of G6Pase activity may be induced by the high level of hydrocortisone at parturition, and furthermore, glucagon may have a permissive effect.     

Activity and isoenzyme patterns of glycolytic enzymes during perinatal development of rat lung

The enzymes hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were studied in rat lung during development starting at day 16 of gestation (day-6) until 5 days after birth. During gestation, the activities of hexokinase type II, enolase and pyruvate kinase decreased and reached adult values at birth or shortly thereafter. Hexokinase type I remained relatively constant and the decrease of soluble type II hexokinase was compensated for by an increment of particle-bound hexokinase starting at day 20 of gestation until birth. In contrast, phosphofructokinase activity increased until day 20 of gestation followed by a rapid fall in activity until 2 days after birth. Except for hexokinase no isoenzyme shifts were observed in the period of observation. The results are discussed with respect to the proposed relationship between glycogen breakdown and surfactant synthesis during the perinatal period and suggest a regulatory role for phosphofructokinase in this process.     

Effects of short-term and prolonged aerogenic hypoxia on gamma-glutamyl transpeptidase activity in the brain,liver, and biological fluids of young rats

Posthypoxic fluctuations in the levels of two excitatory amino acids, glutamate and aspartate, may be related to changes in mechanisms(s) which are responsible for their reuptake. As gamma-glutamyl transpeptidase (GGT) plays a role in mediating the uptake of glutamate and aspartate into various compartments of the brain, we studied changes in the activity of this enzyme in main regions of the brain in young and adult rats. We found a posthypoxic increase in bound GGT activity in some brain regions of 18-day-old animals after acute exposure, but no changes were observed after prolonged altitude hypoxia, with the exception of a decrease in cortical GGT activity. In contrast, acute hypoxia decreased GGT activity in the cortical capillaries to 59%, but prolonged hypoxic exposure was ineffective. However, the activity of soluble GGT in the cerebrospinal fluid of both groups of rats was several-times elevated in comparison with controls. At the same time, bound GGT activity was increased in the liver after acute or prolonged altitude hypoxia. The soluble GGT activity in plasma was only increased after prolonged exposure. Ninety days after prolonged hypoxic exposure the bound GGT activity was reduced in all brain regions to about 60–70% of controls (significantly higher in females than in males) as long-term developmental sequel from early postnatal hypoxia.