Insulin signaling during perinatal liver development in the rat

Insulin has long been assigned a key role in the regulation of growth and metabolism during fetal life. Our prior observations indicated that hepatic insulin signaling is attenuated in the late-gestation fetal rat. Therefore, we studied the perinatal ontogeny of hepatic insulin signaling extending from phosphatidylinositol 3-kinase (PI3K) to the ribosome. Initial studies demonstrated markedly decreased insulin-mediated activation of ribosomal protein S6 kinase 1 (S6K1) in the fetus. We found a similar pattern in the regulation of Akt, a kinase upstream from S6K1. Insulin produced minimal activation of insulin receptor substrate (IRS)-1-associated PI3K activity in fetal liver. A modest IRS-2-associated response was seen in the fetus. However, levels of both IRS-1 and IRS-2 were very low in fetal liver relative to adult liver. IRS-1 content and insulin responsiveness of PI3K, Akt, and S6K1 showed a transition to the adult phenotype during the first several postnatal weeks. Examination of downstream insulin signaling to the translational apparatus showed marked attenuation, relative to the adult, of fetal hepatic insulin-mediated phosphorylation of 4E-BP1, the regulatory protein for the eukaryotic initiation factor eIF4E, and ribosomal protein S6. The mammalian target of rapamycin (mTOR), a key integrator of nutritional and metabolic regulation of translation, was present in low amounts, was hypophosphorylated, and was not insulin sensitive in the fetus. Our results indicate that protein synthesis during late-gestation liver development may be mTOR and insulin independent. Reexamination of the role of insulin in fetal liver physiology may be warranted.     

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.     

Alterations in liver chromatin during perinatal development of the rat

Chromatins were isolated from liver nuclei of 19-day fetuses, 2-, 5-, 21-day old and adult rats. Very little variation was observed in the mass ratio of total histones to DNA or in the spectrum of histones as determined by polyacrylamide gel electrophoresis. On the other hand, the amount and banding pattern of acidic proteins indicated pronounced changes during liver development.The composition of acidic proteins may be specific for the stage of development as evidenced immunochemically. Antibody against acidic protein-DNA complexes from adult rat liver were produced in rabbits. Whereas adult liver acidic protein-DNA complexes interacted strongly with the antibody, fetal liver preparations showed very little affinity. Complexes from 2-day-old animals reacted more strongly than fetal complexes while preparations from 5-day-old and 21-day-old displayed further increases in affinity. The results support the idea that chromatin acidic proteins play an important role in genetic expression during the ontogeny.     

Quantification of surfactant pool sizes in rabbit lung during perinatal development

Methods are presented for the quantitative isolation of surfactants from fetal and newborn rabbit alveolar lavage returns and post-lavaged lung tissue homogenates. The phospholipid content of both fractions progressively increased between 27 days gestation and term (31 days). The tissue-stored fraction increased approximately 16-fold (from 0.48 +/- 0.13 to 7.83 +/- 0.86 mg/g dry lung) and the alveolar fraction more than 30-fold (from 0.08 +/- 0.02 to 2.69 +/- 0.52 mg/g dry lung). Developmental changes in phospholipid composition were also observed. Tissue-stored surfactant was prepared using differential and density gradient centrifugation. Alveolar surfactant was isolated during fetal development as a high-speed pellet following a one-step differential centrifugation. There was little change in the phospholipid content of fetal alveolar lavage supernatant (range 0.12 +/- 0.04 to 0.28 +/- 0.09 mg/g dry lung). By the first postnatal day the phospholipid content of both lavage fractions significantly increased (pellet, 7.51 +/- 1.79; supernatant, 4.01 +/- 1.36 mg/g dry lung) and both were identified as surfactant. This increase in alveolar surfactant was accompanied by an approximately twofold decrease (to 3.81 +/- 1.1 mg/g dry lung) in the tissue-stored fraction. These data provide a quantitative profile of surfactant accumulation and secretion in developing rabbit lung.     

Effect of D-penicillamine on rat lung elastin cross-linking during the perinatal period

This study was designed to clarify the effects of D-penicillamine (DPA), a drug used for treatment of various pathological events, on lung elastin formation and maturation of the newborn in the perinatal period. The investigation was conducted on 20 newborn rats bred from 40 female and six male rats. DPA doses 400 mg kg(-1) day(-1) and physiological saline were given intraperitoneally (i.p) to experimental and control groups. To assess newborn maturation, their body and lung weights were determined. Serum Cu levels were measured by atomic absorption spectroscopy and ceruloplasmin (Cp) activities were measured spectrophotometrically. Newborn lung tissue elastin, desmosine (DES) and isodesmosine (IDES) levels were measured by HPLC. The results showed that DPA treatment caused loss of skin elasticity and reduction in body and lung weight in newborns of the experimental group. The serum Cu levels and Cp activity were found to be significantly lower in both maternal and newborn of the experimental groups compared with the control group. The lung DES, IDES and elastin values of newborns in the experimental group were decreased compared with the control group. In conclusion, our results indicate that 400 mg kg(-1) day(-1) DPA, a dose that is used in the treatment of Wilson's disease, rheumatoid arthritis and cystinuria, caused the retardation of newborn maturation, a decrease in DES-IDES cross-links and levels of lung elastin of offspring in the perinatal period. Another conclusion to be drawn from this study is that even low levels of Cu depletion due to DPA administration induces a change in cross-linking in lung elastin during the perinatal period.     

Levels of mRNAs coding for lipogenic enzymes in rat lung upon fasting and refeeding and during perinatal development

The relative amounts of mRNAs coding for fatty-acid synthase (EC 2.3.1.85), acetyl-CoA carboxylase (EC 6.4.1.2), ATP citrate lyase (EC 4.1.3.8) and malic enzyme (EC 1.1.1.40) were determined in lungs and livers of adult rats that were normally fed, starved for 48 h or starved for 48 h and subsequently refed for 72 h with a carbohydrate-rich, fat-free diet. In the liver, starvation caused a small decrease in the relative abundance of the mRNAs which was not statistically significant. Subsequent refeeding caused a statistically significant increase in mRNAs for all of the enzymes studied. In the lung, no significant changes were found, indicating that the regulation of the abundance of mRNAs encoding the lipogenic enzymes in the lung differs from that in the liver. In the developing rat lung, mRNA for fatty-acid synthase increased 3-fold in abundance between fetal days 18 and 20 and decreased directly after birth (at day 22 of gestation). A similar pattern was observed for ATP citrate lyase mRNA. The level of acetyl-CoA carboxylase mRNA decreased significantly after birth. These observations indicate that in perinatal rat lungs, pretranslational regulation is involved in the control of the synthesis of these enzymes. The abundance of acetyl-CoA carboxylase mRNA did not change in the prenatal period, a time during which the specific activity of this enzyme increases. This lack of correlation between the specific activity of acetyl-CoA carboxylase and the abundance of its mRNA may indicate that translational regulation of the synthesis of the enzyme or post-synthetic regulatory effects on enzyme molecules are involved in the control of this enzyme in the prenatal period. No changes in the abundance of lung malic enzyme mRNAs were observed throughout the perinatal period.     

Albumin and transferrin synthesis during development in the rat

In this study, the incorporation of [(14)C]leucine into albumin and transferrin in early rat foetuses, vitelline plus amniotic membranes, chorioallantoic placenta and perinatal rat liver slices was measured and used to detect and compare the rates of synthesis of the two proteins. Albumin synthesis was detected in the body of foetuses from 13 days gestation onwards. Transferrin synthesis was detected only after day 15. Transferrin synthesis was demonstrable in the membranes but not in the chorioallantoic placenta of all the animals investigated, i.e. from 13 to 19 days gestation. Synthesis of albumin and transferrin by the liver of near-term and postnatal animals was shown to correlate with published data on the parenchymal cell number/unit wet wt. of liver. Near-term foetuses synthesized relatively more transferrin than albumin when compared with 10-day postnatal animals. The serum concentrations of the two plasma proteins were also determined. These increased before term whereas the rate of synthesis of albumin and transferrin declined. Postnatally, plasma albumin concentration increased but transferrin concentration decreased, yet the rates of synthesis of both proteins by the liver increased with age. This lack of correlation between the rates of synthesis of the two proteins and their respective plasma concentrations could be explained in part by their increased stability after birth. There was also evidence that the liver haemopoietic cells took up transferrin although they do not synthesize the protein. Thus the decrease in this population of cells during development could also contribute to the discrepancy between liver synthesis and serum concentrations of transferrin.     

Serotonin content of rabbit lung and small intestine during perinatal development

Serotonin (5-hydroxytryptamine, or 5HT) was measured in extracts of rabbit lung and intestine during perinatal development using high pressure liquid chromatography (HPLC) with electrochemical detection. Lung and intestine were extracted with HClo4 and the extract was loaded onto a Bio-Rex 70 resin column. After elution with acetic acid the samples were injected onto the HPLC column. Serotonin was detected in lung and intestine at 18 days of gestation (80 and 90 ng/mg protein). In lung serotonin content increased at day 28 (290 ng/mg protein) till day 30 (680 ng/mg protein) decreased at day 1 after birth (480 ng/mg protein) and then rose at day 10 of the newborn period (650 ng/mg protein). In intestine the serotonin content was always higher than in the lung. At the end of gestation the serotonin in the intestine remained constant (2410 ng/mg protein at day 28 and 2430 ng/mg protein at day 30), decreased slightly one day after birth (2150 ng/mg protein) and rose at day 10 (3300 ng/mg protein).     

Elastin metabolism in rodent lung

Data from the in vivo incorporation of [³H]valine into fractions of elastin obtained from rat or mouse lung suggest that postnatal lung elastin synthesis occurs predominantly in the first 1 to 2 weeks of life. Very little [³H]valine was incorporated into lung elastin obtained from adult animals. When lung elastin from neonatal mice was radiochemically labelled with [¹⁴C]lysine as a single pulse, it was observed that the specific activity of the elastin expressed as the total dpm values as ¹⁴C per mg was not significantly altered over a 6 month period. Elastin appears to turn over very slowly in mouse lung with half-life best estimated in years.     

Dynamics of apoptosis and proliferation in the rat thymus and spleen during perinatal development

The levels of spontaneous apoptosis and proliferation of the rat thymic and spleen cells, as well as their regulation by the hypothalamo-hypophysial system were studied during perinatal development. The apoptotic and proliferating cells in the thymus and spleen were assayed using flow cytometry with the DNA-specific dye propidium iodide. The level of apoptosis in the thymus reached 25% on day 18 of embryogenesis (E 18) and decreased to 5% thereafter. In the spleen, the level of apoptosis gradually increased from 15 to 37% during the period of E18 to day 30 of postnatal development (P30). The level of dividing cells in the thymus was 20-25% at all developmental stages studied. In the spleen, it was at a maximum on E18 (32%) and decreased almost twice on E21 (17%). On P7, the amount of proliferating cells again increased to 22% and then gradually decreased to 7% by P30. The surgical ablation of hypothalamus in utero on E18 did not affect cell apoptosis or proliferation in the thymus and spleen. The surgical ablation of both hypothalamus and pituitary led a twofold decrease of the level of apoptosis in the spleen and insignificant increase of the level of proliferation in the thymus. Thus, the numbers of cells in the embryonic thymus is regulated not only by the thymus itself, but also by the hypothalamo-hypophysial system. The programmed cell death in the embryonic spleen appears to be regulated by the hypothalamo-hypophysial system as well.     

Changes in lipid synthesis in rat liver during development

1. Lipogenesis, as measured by the incorporation of ¹⁴C-labelled glucose or acetate into fatty acids in liver slices, is high in foetal and adult rat liver but is low in the liver of the suckling rat, especially with glucose as substrate. 2. The rate of synthesis of non-saponifiable lipids from glucose is about 15 times as great in the liver of the 18-day foetus as in adult liver. Activity in the newborn is negligible. 3. Glucose incorporation into fat is strongly concentration-dependent in liver slices from the adult and 2-week-old rat, but less markedly so in liver slices from the foetus. 4. Changes in the activity of hepatic citrate-cleavage enzyme (ATP–citrate lyase) occur in parallel with the changes in the extent of fatty acid formation, supporting the participation of this enzyme in lipogenesis. However, NADP–malate dehydrogenase, a potential source of reduced nucleotide coenzyme for lipogenesis in the adult, could not be detected in foetal rat liver.     

Plasma albumin synthesis during neonatal development of the rat

1. Liver slices were incubated with ¹⁴C-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.     

Mitochondrial component of the phosphorylcreatine shuttle is enhanced during rat heart perinatal development

Aerobic metabolism is enhanced during perinatal heart development in parallel with increased cardiac function. The mitochondrial component of the phosphorylcreatine shuttle is important in providing energy for contraction and was examined in weanling and adult rat left ventricle. Creatine kinase activity was enhanced in tissue homogenate and purified cardiac myocytes of adults. Mitochondrial analyses attribute this enhancement to increased creatine kinase activity per milligram mitochondrial protein. Other enzymatic markers of mitochondrial function are not enhanced in activity during perinatal heart growth. The unique response of creatine kinase points to the shuttle mechanism and of mitochondrial creatine kinase, in particular, as a major contributor to heart functional regulation.     

Regulation of the glucocorticoid receptor in fetal rat lung during development

The effect of the synthetic glucocorticoid betamethasone on the regulation of the glucocorticoid receptor mRNA and on receptor protein was studied in fetal rat lung during development. Using a glucocorticoid receptor cRNA probe, glucocorticoid receptor mRNA was examined by Northern blot hybridization and by solution hybridization. A monoclonal antibody against the glucocorticoid receptor was used to study regulation of the receptor protein by the Western immunoblotting technique. In fetal rat lungs, of 16-21 days of gestation, as well as in adult lungs, betamethasone treatment resulted in a significant decrease of glucocorticoid receptor mRNA to 50-65% of the control level. In contrast, betamethasone treatment did not down-regulate the receptor protein in rat lungs of 16-19 days of gestation, whereas a decrease of glucocorticoid receptor protein to 40-60% of control was seen in lungs of 21 days of gestation, in postnatal and adult lung. These results provide data for a change in regulation in vivo of the glucocorticoid receptor by its homologous ligand in fetal rat lung during development.     

Norepinephrine content of the rat kidney during development: Alterations induced by perinatal methadone

The concentration and the total content of norepinephrine (NE) in the kidney were measured in Sprague-Dawley rats from 3 to 120 days after birth. Renal NE concentration was relatively low until the end of the second week, when it rose abruptly to adult levels; total NE content per kidney increased steadily throughout development. The effects of perinatal methadone treatment on renal NE development were examined by administering the drug either directly to the pups from 1 to 19 days after birth, or to the mother from 10 days of gestation to 20 days after birth. Both treatments resulted in significant deficits of body weight and kidney weight. Maternal methadone caused a significant deficit in renal NE which was most pronounced at two weeks of postnatal age; direct methadone had less effect on renal NE. These results suggest that renal sympathetic neurotransmission may become mature two weeks after birth and indicate further that maternal methadone interfares with this maturation.     

Iroquois genes influence proximo-distal morphogenesis during rat lung development

Lung development is a highly regulated process directed by mesenchymal-epithelial interactions, which coordinate the temporal and spatial expression of multiple regulatory factors required for proper lung formation. The Iroquois homeobox (Irx) genes have been implicated in the patterning and specification of several Drosophila and vertebrate organs, including the heart. Herein, we investigated whether the Irx genes play a role in lung morphogenesis. We found that Irx1-3 and Irx5 expression was confined to the branching lung epithelium, whereas Irx4 was not expressed in the developing lung. Antisense knockdown of all pulmonary Irx genes together dramatically decreased distal branching morphogenesis and increased distention of the proximal tubules in vitro, which was accompanied by a reduction in surfactant protein C-positive epithelial cells and an increase in beta-tubulin IV and Clara cell secretory protein positive epithelial structures. Transmission electron microscopy confirmed the proximal phenotype of the epithelial structures. Furthermore, antisense Irx knockdown resulted in loss of lung mesenchyme and abnormal smooth muscle cell formation. Expression of fibroblast growth factors (FGF) 1, 7, and 10, FGF receptor 2, bone morphogenetic protein 4, and Sonic hedgehog (Shh) were not altered in lung explants treated with antisense Irx oligonucleotides. All four Irx genes were expressed in Shh- and Gli(2)-deficient murine lungs. Collectively, these results suggest that Irx genes are involved in the regulation of proximo-distal morphogenesis of the developing lung but are likely not linked to the FGF, BMP, or Shh signaling pathways.     

Phosphofructokinase in rat lung during perinatal development: characterization of subunit composition and regulation by fructose 2,6-bisphosphate and glucose 1,6-bisphosphate

The subunit composition of phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11) was studied in rat lung during perinatal development. No change in subunit composition during this period was observed. The three subunits of phosphofructokinase (L, M and C) were present in a ratio of approx. 65:25:10, respectively. In addition the levels of two effectors of phosphofructokinase were determined in rat lung during perinatal development: glucose 1,6-bisphosphate and fructose 2,6-bisphosphate. Until day 20 of gestation (term is 22 days) the glucose 1,6-bisphosphate level remains relatively constant (approx. 0.55 mumol/g protein), decreases before birth and increases sharply up to 1.04 mumol/g protein 2 days after birth. The amount of fructose 2,6-bisphosphate in rat lung shows a different developmental profile. A small peak is shown at day 17 of gestation whereas a larger peak up to 36.4 nmol/g protein is shown at days 20 and 21 of gestation. The time of maximal fructose 2,6-bisphosphate content corresponds with the time of glycogen breakdown and acceleration of surfactant synthesis in prenatal rat lung. Both glucose 1,6-bisphosphate and fructose 2,6-bisphosphate stimulate lung phosphofructokinase. Half maximal stimulations occur in the range of 24.1-70.9 microM glucose 1,6-bisphosphate and 0.17-0.34 microM fructose 2,6-bisphosphate.     

Elastin mRNA levels during foetal development of sheep nuchal ligament and lung. Hybridization to complementary and cloned DNA

Elastin mRNA levels were quantified in sheep nuchal ligament and lung during the latter half of foetal development with elastin-specific cDNA (complementary DNA) probes using both hybridization in solution (saturation analysis) and hybridization on a fixed support (Northern analysis). For the solution-hybridization studies, cDNA prepared from nuchal-ligament mRNA was enriched to 65% for elastin sequences by hybridizing it to its template at a R0t (mol X s X litre-1) value that included only the abundant class of mRNA sequences. Hybridization of this probe to RNA extracted from nuchal ligament between 70 and 138 days after conception demonstrated elastin sequences increased about 10-fold (from 0.047 to 0.438% of total RNA). In contrast, lung elastin mRNA levels increased only 3-fold (from 0.009 to 0.022% of total RNA) during the same period. Over this development period these values correspond to increases in the average number of elastin mRNA molecules from 950 to 20 000 molecules/ligament cell and from 130 to 330 molecules/lung cell. For Northern analysis, elastin mRNA was purified from near-term-sheep nuchal ligament on sucrose density gradients. Analysis of the translation products of this elastin mRNA showed that relative elastin precursor synthesis was at least 80% of total [3H]valine incorporation. The Mr of this elastin mRNA, determined by methylmercury-agarose-gel electrophoresis, was approx. 1.25 X 10(6). Northern hybridization of nuchal ligament and lung RNA to a [32P]cDNA probe, transcribed from this sucrose-gradient-purified elastin mRNA, confirmed the developmental changes in elastin mRNA levels detected by solution-hybridization techniques. The specificity of this method was confirmed by using a cloned elastin gene fragment. These studies demonstrate that elastin mRNA levels in organs such as nuchal ligament and lung increase with foetal development, but that there are significant differences in the average cellular elastin mRNA content of these two organs.